AA T AAT Bioquest - INTERCHIM: Home Fluorimetric Fluorescamine Protein Quantitation Kit ..... ..... 10 Amplite Fluorimetric Thiol Quantitation Assay Kit ..... ..... 10 Section 3 ReadiLink

AAT Bioquest, Inc. (Formerly ABD Bioquest, Inc.)923 Thompson PlaceSunnyvale, CA 94085, USATel: 800-990-8053/408-733-1055Fax: 408-733-1304Email: [email protected]

© 2011 by AAT Bioquest, Inc.

2011-2012

Assay Kit Catalog

AAT Bioquest

Labeling Detection ScreeningA

AT

Bio

qu

est Assay

Kit C

atalog

20

11

-20

12

168737j1_CVR.indd 1 2/15/11 10:21 AM

Austria:Biomol GmbHEmail: [email protected]: http://www.biomol.de

Belgium:Gentaur BVBAEmail: [email protected]: http://www.gentaur.com

Canada:Cedarlane Laboratories Ltd.Email: [email protected]: http://www.cedarlanelabs.com

China:Beijing Fanbo Biochemicals Co. Ltd.Email: [email protected]: http://www.fanbobiochemicals.com

Beijing Zhonghao Shidai Co., Ltd (Northern China)Email: [email protected]: http://www.biopcr.com

Dongguan Mag Biotech Co. Ltd (Southern China)Email: [email protected]: http://www.dgmaigen.com

HD Biosciences (China) Co., Ltd (Eastern China)Email: [email protected]: http://www.hdbiosciences.com

Shaanxi Zhendi Chemical Biology Co., Ltd (Western China)Email: [email protected]

Croatia and Czech Republic:Biomol GmbHEmail: [email protected]: http://www.biomol.de

Denmark:Nordic BioSite ApSEmail: [email protected]: http://www.nordicbiosite.dk

Estonia:Biomol GmbHEmail: [email protected]: http://www.biomol.de

Nordic BioSite ABEmail: [email protected]: http://www.biosite.se

Finland:Nordic BioSite OYEmail: [email protected]: http://www.biosite.fi

France:InterchimEmail: [email protected]: http://www.interchim.com

Germany:Biomol GmbHEmail: [email protected]: http://www.biomol.de

Hungary:Biomol GmbHEmail: [email protected]: http://www.biomol.de

IZINTA Trading Co., Ltd.Email: [email protected]: http://www.izinta.hu

Iceland:Nordic BioSite ABEmail: [email protected]: http://www.biosite.se

India:Biochem Life SciencesEmail: [email protected]: http://www.biochemls.in

GenxBio Health Sciences Pvt. Ltd,Email: [email protected]: [email protected]: http://www.genxbio.com

Israel:Biological Industries Ltd.Email: [email protected]: http://www.bioind.com

Italy:Space Import Export S.r.l.Email: [email protected]: http://www.spacesrl.com

Japan:Cosmo Bio Co., Ltd.Email: [email protected]: http://www.cosmobio.co.jp

Nacalai Tesque, Inc.Email: [email protected]: http://www.nacalai.com

Wako Pure Chemical Industries, Ltd.Email: [email protected]: http://www.wako-chem.co.jp

Korea:Cheong Myung Science CorporationEmail: [email protected]: http://www.cmscorp.co.kr

Latvia and Lithuania:Nordic BioSite ABEmail: [email protected]: http://www.biosite.se

Netherlands:Gentaur BVBAEmail: [email protected]: http://www.gentaur.com

InterchimEmail: [email protected]: http://www.interchim.com

Norway:Nordic BioSite ABEmail: [email protected]: http://www.biosite.se

Poland, Slovakia and Slovenia:Biomol GmbHEmail: [email protected]: http://www.biomol.de

Romania:SC VitroBioChem SRLEmail: [email protected]: http://www.vitro.ro

Singapore and Other South Asian Countries:BST Scientific Pte LtdEmail: [email protected]: http://www.bstsci.com

Curiox Biosystems Pte LtdEmail: [email protected]: http://www.curiox.com

South American Countries and Regions:Impex Comércio Internacional LtdaEmail: [email protected]: http://www.impexbrasil.com.br

Spain:Gentaur BVBA Email: [email protected]: http://www.gentaur.com

InterchimEmail: [email protected]: http://www.interchim.com

Sweden:Nordic BioSite ABEmail: [email protected]: http://www.biosite.se

Switzerland:LuBioScience GmbHEmail: [email protected]: http://www.lubio.ch

Taiwan:Rainbow Biotechnology Co., LTD.Email: [email protected]: http://www.rainbowbiotech.com.tw

Turkey:Biomol GmbHEmail: [email protected]: http://www.biomol.de

United Kingdom:Stratech Scientific LtdEmail: [email protected]

International Distributors

Our Mission

AAT Bioquest is committed to constantly meet or exceed its customer’s requirements by providing consis-tently high quality products and services and by encouraging continuous improvements in its long-term and daily operations. Our core value is Innovation and Customer Satisfaction.

Our Story

AAT Bioquest, Inc. (formerly ABD Bioquest, Inc.) develops, manufactures and markets bioanalytical research reagents and kits to life sciences research, diagnostic R&D and drug discovery. We specialize in photometric detections including absorption (color), fluorescence and luminescence technologies. The Company's superior products enable life science researchers to better under-stand biochemistry, immunology, cell biology and molecular biology. AAT Bioquest offers a rapidly expanding list of enabling products. Besides the standard catalog products we also offer custom service to meet the distinct needs of each customer. Our current services include custom synthesis of biological detection probes, custom development of biochemical, cell-based and diagnostic assays and custom high throughput screening of drug discovery targets.

It is my greatest pleasure to welcome you to AAT Bioquest. We greatly appreciate the constant support of our valuable customers. While we continue to rapidly expand, our core value remains the same: Innovation and Customer Satisfaction. We are committed to being the leading provider of novel biological detection solutions. We promise you to extend these values to you during the course of our service and to continue to support you with our new products and services. It is our greatest honor to receive valu-able feedbacks and suggestions from you so that we can better serve your projects.

Very truly yours,

Zhenjun Diwu, Ph.D.President

168737j1_CVR.indd 2 2/15/11 10:21 AM

1

Table of Contents

Section 1 General information 1

Section 2 Protein Analysis and Modification 5

Amplite™ Colorimetric Biotin Quantitation Kit ..................................................................................... 7ReadiLink™ Protein Biotinylation Kit .................................................................................. ................. 7ReadiLink™ BSA Conjugation Kit .......................................................................................................... 8ReadiLink™ KLH Conjugation Kit........................................................................................... ............... 8Amplite™ Colorimetric Maleimide Quantitation Kit............................................................... ............... 9Amplite™ Fluorimetric Maleimide Quantitation Kit ............................................................... .............. 9Amplite™ Fluorimetric Fluorescamine Protein Quantitation Kit ........................................... .............. 10Amplite™ Fluorimetric Thiol Quantitation Assay Kit ............................................................ .............. 10

Section 3 Protein Labeling with Fluorescent Dyes 11

ReadiLink™ iFluor™ 488 Protein Labeling Kit.................................................................... .................. 13ReadiLink™ iFluor™ 555 Protein Labeling Kit.................................................................... .................. 13ReadiLink™ iFluor™ 594 Protein Labeling Kit.................................................................... .................. 14ReadiLink™ iFluor™ 647 Protein Labeling Kit.................................................................... .................. 14ReadiLink™ iFluor™ 680 Protein Labeling Kit.................................................................... .................. 15ReadiLink™ iFluor™ 700 Protein Labeling Kit ................................................................... .................. 15ReadiLink™ iFluor™ 750 Protein Labeling Kit.................................................................... .................. 16ReadiLink™ iFluor™ 780 Protein Labeling Kit ................................................................... .................. 16ReadiLink™ mFluor™ Violet 420 Protein Labeling Kit ....................................................... .................. 17ReadiLink™ mFluor™ Violet 450 Protein Labeling Kit....................................................... ................... 17 ReadiLink™ mFluor™ Violet 520 Protein Labeling Kit...................................................... .................... 18ReadiLink™ mFluor™ Violet 560 Protein Labeling Kit ...................................................... .................. 18

Amplite™ Fluorimetric Acetylcholine Assay Kit................................................................ ..................21PhosphoWorks™ Fluorimetric ADP Assay Kit.......................................................................................21Amplite™ Colorimetric Aldehyde Quantitation Kit........................................................... ...................22Amplite™ Fluorimetric Aldehyde Quantitation Kit............................................................ ..................22PhosphoWorks™ Luminometric ATP Assay Kit ................................................................ ...................23Amplite™ Calcium Quantitation Kit ................................................................................. ..................24Amplite™ Cholesterol Quantitation Kit........................................................................... ....................24Amplite™ Choline Quantitation Kit................................................................................... ..................25Amplite™ Ethanol Quantitation Kit................................................................................... ..................25Amplite™ Glucose Quantitation Kit.................................................................................. ...................26Amplite™ Fluorimetric Glutamic Acid Assay Kit............................................................... ...................26Amplite™ Fluorimetric Glutathione Assay Kit ................................................................. ...................27 Amplite™ Fluorimetric Glutathione GSH/GSSG Ratio Assay Kit .......................................... ................27Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit........................................................ ..................28Amplite™ Colorimetric NAD/NADH Assay Kit.................................................................... ..................29Amplite™ Fluorimetric NAD/NADH Assay Kit ................................................................... ..................29Amplite™ Fluorimetric NAD/NADH Ratio Assay Kit.......................................................... ...................30Amplite™ Fluorimetric NADH Assay Kit........................................................................... ....................30Amplite™ Colorimetric NADP/NADPH Assay Kit............................................................... ...................31Amplite™ Fluorimetric NADP/NADPH Assay Kit................................................................ ..................31Amplite™ Fluorimetric NADP/NADPH Ratio Assay Kit ...................................................... ..................32Amplite™ Fluorimetric NADPH Assay Kit ......................................................................... ...................32PhosphoWorks™ Colorimetric MESG Phosphate Assay Kit...................................................................33PhosphoWorks™ Colorimetric Phosphate Assay Kit.......................................................... ..................33PhosphoWorks™ Fluorimetric Phosphate Assay Kit ............................................................................34PhosphoWorks™ Fluorimetric Pyrophosphate Assay Kit................................................... ..................34

Section 5 Enzymatic Activity Analysis 35

Amplite™ Colorimetric Acetylcholinesterase Assay Kit.........................................................................37Amplite™ Fluorimetric Acetylcholinesterase Assay Kit................................................................... 37-38Amplite™ Colorimetric Alkaline Phosphatase Assay Kit................................................. ......................37Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit .............................................. .................. 39-40Amplite™ Luminometric Alkaline Phosphatase Assay Kit................................................ ....................40Amplite™ Fluorimetric Caspase 3/7 Assay Kit .................................................................. ...................41Amplite™ Fluorimetric Catalase Assay Kit ........................................................................ ...................42Amplite™ Fluorimetric Glucose Oxidase Assay Kit............................................................. ...................42Amplite™ Fluorimetric Glutamate Oxidase Assay Kit........................................................ ...................43Amplite™ Fluorimetric HDAC Activity Assay Kit ................................................................ ...................43Amplite™ Universal Fluorimetric Kinase Assay Kit............................................................ ...................44Amplite™ Fluorimetric Lysyl Oxidase Assay Kit ................................................................ ...................44Amplite™ Fluorimetric Monoamine Oxidase Assay Kit..................................................... ....................45Amplite™ MMP-3 Activity Assay Kit ................................................................................. ...................45

Section 4 Quantification of Biochemical Molecules 19

Amplite™ Universal Fluorimetric MMP Activity Assay Kit................................................ ................... 46Amplite™ Fluorimetric Myelopeoxidase Assay Kit............................................................ .................. 47ReadiUse™ NADPH Regenerating Kit................................................................................ .................. 47Amplite™ Colorimetric Peroxidase Assay Kit .................................................................... .................. 48Amplite™ Fluorimetric Peroxidase Assay Kit................................................................ ..................48-49Amplite™ Luminometric Peroxidase Assay Kit.................................................................. .................. 49Amplite™ Fluorimetric Goat Anti-Mouse IgG-HRP Conjugate ELISA Assay Kit.................. ................... 50Amplite™ Fluorimetric Goat Anti-Rabbit IgG-HRP Conjugate ELISA Assay Kit.................. ................... 50Amplite™ Fluorimetric Proteasome 20S Activity Assay Kit............................................... ................... 51Amplite™ Universal Fluorimetric Protease Activity Assay Kit...................................... ...................51-52Amplite™ Fluorimetric Renin Assay Kit........................................... ................................ ................... 52Amplite™ Colorimetric Sphingomyelinase Assay Kit........................................................ ................... 53Amplite™ Fluorimetric Sphingomyelinase Assay Kit........................................................ ................... 53Amplite™ Colorimetric Superoxide Dismutase (SOD) Assay Kit........................................ ................... 54Amplite™ Fluorimetric Xanthine Oxidase Assay Kit ......................................................... ................... 54

FluoroQuest™ Anti-fading Kit I .......................................................................................................... 57FluoroQuest™ Anti-fading Kit II ......................................................................................................... 57Cell Explorer™ Fixable Dead Cell Labeling Kit ................................................................................58-61Cell Explorer™ Live Cell Labeling Kit .............................................................................................62-64Cell Explorer™ Cell Tracking Kit .....................................................................................................65-66Cell Navigator™ F-Actin Labeling Kit .............................................................................................67-68Cell Navigator™ Lysosomal Staining Kit ........................................................................................69-70Cell Navigator™ Mitochondrial Staining Kit ..................................................................................71-72

Section 6 Cell Labeling and Tracking 55

Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit ........................................................................ 75Cell Meter™ Fluorometric Caspase 3/7 Activity Assay Kit ................................................................... 76Cell Meter™ Generic Fluorometric Caspase Activity Assay Kit ........................................................76-77Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit ........................................................................... 77Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit ............................................................................78Cell Meter™ Intracellular GSH Assay Kit ............................................................................................. 78Cell Meter™ Nuclear Apoptosis Assay Kit ........................................................................................... 79Cell Meter™ Phosphatidylserine Apoptosis Assay Kit ....................................................................79-83Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit ............................................................... 83Screen Quest™ Fluo-8 No Wash Calcium Assay Kit .............................................................................. 84Screen Quest™ Luminometric Calcium Assay Kit................................................................................. 84Screen Quest™ Ratiometric Fura-2 No Wash Calcium Assay Kit ........................................................... 85Screen Quest™ Rhod-4 No Wash Calcium Assay Kit .......................................................................85-86Screen Quest™ Colorimetric ELISA cAMP Assay Kit.............................................................................. 86Screen Quest™ Fluorimetric ELISA cAMP Assay Kit .............................................................................. 87Screen Quest™ Fluorimetric No Wash cAMP Assay Kit ........................................................................ 87Screen Quest™ FRET No Wash cAMP Assay Kit .................................................................................... 88Screen Quest™ Live Cell cAMP Assay Serive Pack ................................................................................ 88Cell Meter™ Cell Viability Assay Kit ...............................................................................................89-90Cell Meter™ Fluorimetric Cell Cycle Assay Kit ..................................................................................... 91Cell Meter™ Colorimetric Cell Cytotoxicity Assay Kit ............................................................................ 92Cell Meter™ Fluorimetric Cell Cytotoxicity Assay Kit ............................................................................ 92Screen Quest™ Colorimetric Chloride Channel Assay Kit ..................................................................... 93Amplite™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit ..................................................... 93Screen Quest™ Fluorimetric MDR Assay Kit ........................................................................................ 94Screen Quest™ Membrane Potential Assay Kit ................................................................................... 94Cell Meter™ JC-10 Mitochondrial Membrane Potential Assay Kit ....................................................... 95Cell Meter™ Mitochondrial Membrane Potential Assay Kit ................................................................ 96Cell Meter™ NIR Mitochondria Membrane Potential Assay Kit ........................................................... 97Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit .................................................... 98

Section 7 Cellular Function Analysis 73

Section 8 Reporter Gene Analysis 99

Amplite™ Fluorimetric Beta-Galactosidase Assay Kit ....................................................................... 101Amplite™ Gaussia Luciferase Reporter Gene Assay Kit ..................................................................... 101Amplite™ Luciferase Reporter Gene Assay Kit .................................................................................. 102Amplite™ Renilla Luciferase Reporter Gene Assay Kit ...................................................................... 102

Section 9 Index 103

Keyword Index ................................................................................................................................. 104Alphabetical Index ........................................................................................................................... 105Catalog Number Index ..................................................................................................................... 107

168737j1_cat.indd 1 2/4/11 11:51 AM

General Information www.aatbio.com

Tel: 800-990-8053 • Fax: [email protected] • [email protected]

Gen

eral

Info

rmat

ion

Unless otherwise specified, all products are for Research Use Only. Not for use in diagnostic or therapeutic procedures.

1

2

Trademarks of AAT Bioquest

AAT Bioquest®Amplite™ Apopxin™Calcein Green™Calcein Orange™Calcein Red™Cell Explorer™ Cell Navigator™ Cell Meter™ CytoCalcein™FP Fluor™ HDAC Green™iFluor™ Iodide Blue™mFluor™MitoLite™MMP Green™MMP Red™NIR Fluor™ Nuclear Green™Nuclear Orange™Nuclear Red™PhosphoWorks™ Prolite™ Quest Fluo-8™Quest Rhod-4™Quest View™ ReadiView™ RatioWorks™ ReadiLink™ReadiUse™Screen Quest™ StainIt™SunRed™

Trademarks of Other Companies

Alamar Blue® (AccuMed International)Alexa Fluor® (Invitrogen)Cy3®, Cy5®and Cy7® (GE Healthcare)DyLight™ (ThermoFisher)FDSS™ (Hamamatsu Corporation)FLIPR® (Molecular Devices)IRDye® 700 and IRDYE 800® (LI-COR)Pacific Blue® and Pacific Orange® (Invitrogen)Texas Red®(Invitrogen)

Thiolite™Tide Fluor™ Tide Quencher™TR Fluor™ VFSE™

www.aatbio.com General Information

General Inform

ation


Unless otherwise specified, all products are for Research Use Only. Not for use in diagnostic or therapeutic procedures. 3

1

CUSTOMER SERVICE & ORDERING INFORMATION

AAT Bioquest Corporate Headquarter:AAT Bioquest, Inc. (Formerly ABD Bioquest, Inc.)923 Thompson PlaceSunnyvale, CA 94085, USATel: 800-990-8053 (US) 408-733-1055 (International)Fax: 408-733-1304

Website: www.aatbio.com

e-mails: [email protected] (inquire)

[email protected] (quote request)

[email protected] (technical support)

International Distributors:

See Back Cover

TERMS AND CONDITIONS OF SALE

1. Prices, Orders and Changes: Prices shown are in US currency. Please call us for current prices if you require this information prior to placing your order. We guarantee our written quotations for 60 days. You may not cancel purchase orders unless such cancellation is expressly agreed by us. In such event, you will be advised of the total charge for such cancellation. You agree to pay such charges, including, but not limited to, storage and shipment costs, costs of producing non-standard materials, costs of purchasing non-returnable materials, cancellation costs imposed on us by our suppliers, and any other cost resulting from cancellation of this order.

2. Delivery: In most cases, we use standard overnight or two-day Federal Express delivery (or equivalent). All shipping charges billed are the responsibility of the customer and are normally prepaid by AAT Bioquest, Inc. and added to the invoice. We reserve the right to make delivery in installments, all such installments to be separately invoiced and paid for when due per invoice, without regard to subsequent deliveries. Partial shipments of available items are made when another item is backordered. Please inspect your packages upon receipt. If the goods have been damaged in transit, we can assist you in filing a claim with the carrier. You shall notify us in writing of any claims for shortages, defects or damages and shall hold the goods for our written instructions concerning disposition. Any claims for such errors must be made within 10 business days. If it is our error, we will do whatever is necessary to ship the correct products as soon as possible. If you shall fail to notify us any defects within 10 days after the goods have been re-ceived, such goods shall conclusively be deemed to conform to the terms and conditions hereof and to have been irrevocably accepted by the buyer.

3. Payment: Terms of sale are net 30 days of date of invoice that is sent to you within 24 hours of shipping the order. The amount received must be sufficient to cover both the invoiced amount and any bank charges that may be incurred. Late charges may be added to invoices not paid within the 30-day time period. Late charges must be paid before subsequent orders can be shipped.

4. Warranties: The products shipped by AAT Bioquest are warranted to conform to the chemical or biological descriptions provided in our publications. This warranty is exclusive, and we makes no other warranty, express or implied, including any implied warranty of merchantability or fitness for any particular purpose. Our sole and exclusive liability and your exclusive remedy with respect to products proved to our satisfaction to be defective or nonconforming shall be replacement of such products without charge or refund of the purchase price, in our sole discre-tion, upon the return of such products in accordance with our instructions. We will not be liable for any incidental, consequential or contingent damages involving their use.

5. Returns: We must authorize any returns. We will not accept return shipments unless we have given prior written permission and shipping instructions. Goods may not be returned for credit except with our permission, and then only in strict compliance with our return shipment instruc-tions. Any returned items may be subject to a 20% restocking fee. In many cases, items ordered in error cannot be returned because of the sensitive nature of many of our products and the difficulty and expense of requalifying returned items. If items are accepted for return, they must be in new, unopened, unused and undamaged condition, and you will be charged a per-unit 20% restocking charge.

6. Use of Our Products: Our products are used ONLY for laboratory research and development purposes. We realize that, since our products are, unless otherwise stated, intended primarily for research purposes, they may not be on the Toxic Substances Control Act (TSCA) inventory. You assume responsibility to assure that the products purchased from us are approved for use under TSCA, if applicable. You have the responsibility to verify the hazards and to conduct any further research necessary to learn the hazards involved in using products purchased from us. You also have the duty to warn your customers and any auxiliary personnel (such as freight handlers, etc.) of any risks involved in using or handling the products.

7. Patent Disclaimer: We do not warrant that the use or sale of our products will not infringe the claims of any United States or other patents covering the product itself or the use thereof in combination with other products or in the operation of any process.

8. Miscellaneous: We reserve the right to discontinue our products or change specifications or prices of our products and to correct any errors or omissions at any time without incurring obligations.

General Information www.aatbio.com


Gen

eral

Info

rmat

ion

Unless otherwise specified, all products are for Research Use Only. Not for use in diagnostic or therapeutic procedures.4

1

Custom Products and Services

Our Technologies

Amplite™ enzyme-based detection platform is optimized for measuring horseradish peroxidase (HRP), alkaline phosphates, luciferase, beta-galactosidase, lactamase, oxidase, protein kinases, protein phosphatases, phosphodiesterases, proteases, cytochrome P450, histone deacetylase (HDAC) and cell signal-ing molecules such as NAD/NADH, NADP/NADPH, IP3, cAMP and cGMP etc.

Cell Explorer™ cell labeling platform is a complete set of tools for tracking live cells. This platform is also widely used for sort-ing mixed populations of cells.

Cell Navigator™ cell staining platform is a complete set of tools for selective labeling subcellular structures of live, fixed and dead cells.

Cell Meter™ cellular functional assay platform is a complete set of tools for functional analysis of cellular events and real time-monitoring of cell functions.

iFluor™ superior fluorescent labeling dyes are optimized for labeling proteins and nucleic acids. This group of dyes span from UV to infrared wavelength with good photostability and brightness.

NIR Fluor™ superior fluorescent labeling dyes are optimized for in vivo imaging. Our NIR Fluor™ 850 is the dye that per-forms best in the longest excitation and emission wavelength.

PhosphoWorks™ detection platform is a set of tools for detec-tion of ATP, ADP, AMP, phosphate, pyrophosphate, phospho-proteins and phosphopeptides.

Quest View™ colorimetric protease platform is a sensitive and robust tool for rapid detection of protease and glycosidase biomarkers. This technology platform has been licensed by a few diagnostic companies for developing rapid diagnostic tests.

RatioWorks™ superior cellular dyes are a sensitive and robust tool set for ratio imaging and real time monitoring of cellular functions (such as pH and ions) in live cells.

Screen Quest™ assay kits are a set of HTS-ready tools for high throughput screening of biochemical and cellular targets such as protein kinases, proteases, HDAC, cell apoptosis and cyto-toxicity, GPCR, ion channels, ADME and transporters.

Our Services

Besides the standard assay kits described in this catalog we also offer custom services to meet the distinct needs of each customer. Our current services include custom synthesis of biological detection probes, custom development of bio-chemical, cell-based and diagnostic assays and custom high throughput screening of drug discovery targets.

Custom Synthesis of Fluorophores and LuminophoresAAT Bioquest is recognized by the top pharmaceutical com-panies and diagnostic companies as a key provider of novel fluorescent dyes and luminescent probes. Over the years we have synthesized many enabling fluorescent and luminescent probes for running a variety of challenging biological detec-tion tasks.

Assay Design and DevelopmentAt AAT Bioquest we not only make probes and assay kits, but also use them extensively ourselves. Scientists at AAT Bioquest are experts at assay design and we have developed a wide va-riety of tests that range from biochemical detection to cellular fuctions. Our assay options include:

• Biochemical molecules • Enzyme activities • Binding assays • Cellular events • Microplate assays • Flow cytometric analysis • Fluorescence imaging • Microarray analysis

Custom Screening AAT Bioquest offers on-demand high-throughput screening and pharmacology profiling assays with multiple methodolo-gies. Functional assays are designed, validated and customized to the needs of our pharmaceutical and biotechnology indus-try clients. These assays are aimed at assessing and monitoring the efficacy, tolerability and safety parameters of candidate compounds for treating and/or diagnosing cancer, infectiousdisease, autoimmunity and transplantation. Our screeningoptions include:

Tide Fluor™ and Tide Quencher™ superior labeling dyes are specially optimized for labeling nucleotides and peptides. This platform offers the best value in the industry. It is second to none in regarding to performance and cost. This technology platform has been licensed by a few diagnostic companies for developing IVD diagnostic tests.

TR Fluor™ superior fluorescent labeling dyes are optimized for developing time-resolved fluorescence-based assays. It has been used for developing HTS assay technologies for many drug discovery targets.

• Full assay development for a target of your choice • Optimization of your assay protocol for HTS • High throughput screening/profiling with significant economies of scale for large compound numbers • Rapid testing of lead compound analogue libraries or screening hits for multi-parametric activity profiles (primary assay potency, in vitro ADMET, selectivity) • Multiple assay platforms/detection methods • Custom data analysis

168737j1_cat.indd 4 2/4/11 11:51 AM

Tel: 800-990-8053 • Fax: [email protected] • [email protected] 5

Protein Analysis and Modification

Protein Analysis and M

odification

2

168737j1_cat.indd 5 2/4/11 11:51 AM



Protein Analysis and Modification www.aatbio.com P

rote

in A

naly

sis

and

Mod

ifica

tion

2protein chemistry kits at-a-glance*

Protein Tools Absorption Fluorescence Other

Biotin Quantitation 5522

Biotinylation 5521

BSA Conjugation 5501

KLH Conjugation 5502

Maleimide Quatitation 5523

Protein Quantitation 11100

Thiol Quantitation 5524

* Products listed by catalog number

168737j1_cat.indd 6 2/4/11 11:51 AM



2

Biotin Quantitation/Biotinylation

ReadiLinkTM Protein Biotinylation Kit * Powered by ReadiViewTM Biotin Visionization Technology*

Cat # Size Storage Condition5521 1 kit < - 15 oC

Biotin is widely used for labeling biomolecules, in particular, antibodies. This kit is primarily optimized for the preparation of biotin-labeled antibodies for enzyme immunoassay. However, it can also be used to label any proteins and biomolecules that contain primary amines. The kit uses our ReadiView™ Biotin (3059) that reacts with an amino group of antibodies and other biomolecules. Our unique biotin contained in the kit carries a color tag to indicate the degree of biotinylation, thus eliminating the troublesome HABA biotinylation determination step. The HABA biotinylation assay is notoriously inaccurate although many efforts have been taken to improve the HABA assay accuracy (including our kit 5522). The color tag is carefully selected to avoid the interference of either biotin binding or fluorescence detection.

This kit contains all the necessary reagents for labeling and purifica-tion. On our hands, 5 to 8 biotin molecules can be conjugated to each IgG molecule using this kit. The kit is designed for 3 conjuga-tion reactions. For each conjugation reaction the amount of mate-rial can label up to 10 mg protein. The entire process only takes less than an hour. The degree of biotinylation can be readily calculated by the following equation with a simple absorption spectrum.

Number of Biotin/Conjugate = [A351/29300] ÷ [A280/ξprotein]

Protein Analysis and Modificationwww.aatbio.com

AmpliteTM Colorimetric Biotin Quantitation Kit


The avidin/streptavidin-biotin interaction is the strongest known non-covalent biological interaction (Kd = 10-15 M-1) between a pro-tein and its ligand. The bond formation between biotin and avidin/streptavidin is very rapid and unaffected by pH, organic solvents, and other denaturing agents. Both avidin and streptavidin have essentially irreversible biotin-binding properties since bound biotin can only be released by denaturing the subunits of the proteins. The tight and specific binding of biotin and its derivatives to various avidins has been extensively explored for a number of biological applications.

Amplite™ Colorimetric Biotin Quantitation Kit is used for determin-ing the molar ratio of biotin incorporated into a protein using the HABA-Avidin method. The HABA dye (4´-hydroxyazobenzene-2-car-boxylic acid) binds to avidin to produce a yellow-orange colored complex which absorbs at 500 nm. Free biotin will displace the HABA dye and cause the absorbance to decrease. A standard curve can be established using the free biotin to estimate the number of moles of biotin incorporated after biotinylating a protein. The kit provides a convenient method for estimating the molar ratio of biotin to protein in biotin-protein conjugates or for quantitating biotin concentration in a solution. It is best used to determine biotin concentration in the range from 1 to 20 µM.

Related Products

Cat # Size Product Name

3002 100 mg Biotin, succinimidyl ester

3010 25 mg Biotin-X, succinimidyl ester

3050 25 mg ReadiViewTM Biotin acid

3053 5 mg ReadiViewTM Biotin amine

3055 5 g ReadiViewTM Biotin hydrazide

3058 5 mg ReadiViewTM Biotin maleimide

3059 5 mg ReadiViewTM Biotin succinimidyl ester

Figure 2.1. HABA Assay principle for quantifying biotinylation degree. Figure 2.2. The chemical structure of ReadiViewTM biotin.

CT=Color Tag, RM=Reactive Moiety, SP=Spacer



Protein Analysis and Modification www.aatbio.com

2

BSA

/KLH

Con

juga

tion

ReadiLinkTM KLH Conjugation Kit *For Antibody Development*

Cat # Size Storage Condition5502 1 kit 2-6 oC

Keyhole Limpet Hemocyanin (KLH) is one of the most commonly used carrier proteins in the conjugation of peptides for antibody production. Mariculture keyhole limpet hemocyanin (mcKLH) is a hemocyanin from the Concholepas concholepas mollusk with immunogenic properties similar to those of KLH. However, mcKLH is a more stable and efficient carrier protein for the production of antibodies to haptens and peptides. It contains numerous sites per molecule for effective conjugation of peptides and other antigens using amine-reactive or carboxy-reactive crosslinkers. mcKLH is currently the industry standard for antibody production against a hapten or peptide. This ReadiLink™ KLH Conjugation Kit is primarily optimized for the simple preparation of hapten-carrier conjugates for immunization and antibody production.

Our ReadiLink™ KLH Conjugation Kit provides a one-step conjuga-tion method of a hapten to a carrier protein using the carboxy-reac-tive carbodiimide as the crosslinker. The resulting conjugate is used for eliciting an immune response and antibody production against the hapten. The carboxy-reactive carbodiimide reacts with exposed carboxy and amino groups on peptides and proteins to form stable bonds. The kit contains mcKLH formulated in buffers compatible with the carboxy-reactive carbodiimide reactions and desalting spin columns, which offer exceptional protein recovery with a simple centrifugation step.

ReadiLinkTM BSA Conjugation Kit *For Antibody Development*


Bovine serum albumin (BSA) is the most abundant protein used for numerous biochemical applications including ELISAs (Enzyme-Linked Immunosorbent Assays), immunoblots, and immunohisto-chemistry. Like most abundant plasma proteins, BSA is very stable and soluble. In addition, the 67 kDa protein is sufficiently large and complex to be fully immunogenic. It contains numerous sites per molecule for effective conjugation of peptides and other antigens using amine-reactive or carboxy-reactive crosslinkers. Consequent-ly, BSA is a popular carrier protein for conjugation to haptens and other weak antigens to make them more immunogenic for antibody production. This ReadiLink™ BSA Conjugation kit is primarily opti-mized for the simple preparation of hapten-carrier conjugates for immunization and antibody production.

Our ReadiLink™ BSA Conjugation Kit provides a one-step conjuga-tion method of a hapten to a carrier protein using the carboxy-reac-tive carbodiimide as the crosslinker. The resulting conjugate is used for eliciting an immune response and antibody production against the hapten. The carboxy-reactive carbodiimide reacts with exposed carboxy and amino groups on peptides and proteins to form stable bonds. The kit contains BSA formulated in buffers compatible with the carboxy-reactive carbodiimide reactions and desalting spin columns, which offer exceptional protein recovery with a simple centrifugation step.

Related Products


5522 1 kit AmpliteTM Colorimetric Biotin Quantitation Kit

4507 100 mg 3-Maleimidopropionic acid N-hydroxysuccinimide ester

5521 1 kit ReadiLink™ Protein Biotinylation Kit *Powered by ReadiView™ Biotin Visionization Technology*

4501 25 mg SMCC [4-(N-Maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester]

4502 1 g SMCC [4-(N-Maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester]

4503 5 mg SMCC Plus™ *Enhanced water solubility, crosslinking efficiency and stability*

4505 25 mg Sulfo-SMCC [4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester, sodium salt]

Figure 2.3. EDC reacts with a carboxyl group of carrier protein BSA or KLH (represented by the red ball), forming an amine-reactive O-acylisourea intermediate (the central molecule). The O-acylisourea intermediate reacts with an amine group on the antigen molecule represented by the smaller blue ball, yielding a conjugate of the two molecules joined by a stable amide bond [Please note the O-acylisourea intermediate is also susceptible to hydrolysis, making it unstable and short-lived in aqueous solution].

168737j1_cat.indd 8 2/4/11 11:51 AM



www.aatbio.com Protein Analysis and Modification

2

Maleim

ide Quantitation

AmpliteTM Fluorimetric Maleimide Quantitation Kit*Green Fluorescence*


Sensitive assays of maleimide and thiol groups are required for the efficient conjugation of proteins that are expensive and available only in small amounts. A variety of crosslinking reagents with a ma-leimide group are widely used for crosslinking proteins to proteins or proteins to other biomolecules. There are few reagents or assay kits available for quantifying the number of maleimide groups that are introduced into the first protein. All the commercial kits have tedious protocols.

Our Amplite™ Fluorimetric Maleimide Qutitation kit uses a pro-prietary dye that has enhanced fluorescence upon reacting with a maleimide. The kit provides a sensitive, one-step fluorimetric method to detect as little as 10 picomoles of maleimide in a 100 µL assay volume (100 nM). The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to au-tomation without a separation step. Its signal can be easily read by a fluorescence microplate reader at Ex/Em = 490/520 nm. Compared to kit 5525, this fluorometric assay is more sensitive, and has less interference from biological samples.

Amplite™ Colorimetric Maleimide Quantitation Kit


Maleimides can be directly assayed spectrophotometrically at 302 nm. However, the small extinction coefficient of 620 M-1cm-1 renders this assay insensitive, and the assay is further complicated by the protein absorbance at the same wavelength.

This colorimetric maleimide assay kit quantifies maleimide groups by first reacting a sample with a known amount of thiol present in excess and then assaying the remaining unreacted thiol using 4,4'-DTDP with a molar extinction coefficient of 19,800 M -1cm-1. The amount of maleimide is calculated as the difference between the initial amount of thiol and the amount of unreacted thiol after the complete reaction of all maleimide groups. This spectrophotometric assay for the determination of maleimide groups is a reverse GSH assay. It takes advantage of the high reactivity of thiols of GSH with the maleimide moiety. Maleimide of the sample is allowed to form a stable thiosuccinimidyl linkage with GSH. After the reaction of the sample is complete, the excess GSH, i.e., the remaining thiols of GSH in the reaction mixture, is estimated by using 4,4'-DTDP. The amount of GSH reacted with the sample is titrated to determine the extent of maleimide.

Related Products


634 5 mg bBBr [Dibromobimane] *UltraPure Grade*

633 25 mg mBBr [Monobromobimane] *UltraPure Grade*

635 10 mg mBCl [Monochlorobimane] *UltraPure Grade*

820 25 mg Fluorescamine *UltraPure Grade*

825 25 mg NBD-Cl [4-Chloro-7-nitrobenzofurazan] *UltraPure grade*

821 5 mg NBD-F [4-Fluoro-7-nitrobenzofurazan] *UltraPure grade*

21508 5 mg Thiolite™ Green

Figure 2.5. N-ethylmaleimide dose response was measured in a 96-well black solid plate with Amplite™ Fluorimetric Maleimide Quantitation Assay Kit using a NOVOStar microplate reader (BMG Labtech). As low as 0.1 µM (10 picomol/well) of maleimide can be detected with 10 minutes incuba-tion time (n=3).

Figure 2.4. 4,4'-DTDP Assay principle for quantifying Malemide.

Strong absorption @ 324 nm

168737j1_cat.indd 9 2/4/11 11:51 AM



Protein Analysis and Modification www.aatbio.com

2

Prot

ein/

Thio

l Qua

ntita

tion

Amplite™ Fluorimetric Thiol Quantitation Assay Kit*Green Fluorescence*


The detection and measurement of free thiol (such as free cysteine, glutathione, and cysteine residues in proteins) is one of the essential tasks for investigating biological processes and events in many bio-logical systems. There are a few reagents or assay kits available for quantitating thiol content in biological systems. All the commercial kits either lack sensitivity or have tedious protocols.

Our Amplite™ Fluorimetric Thiol Qutitation Assay Kit provides an ultrasensitive fluorimetric assay to quantitate thiol content that exists either in a small molecule or on a protein. The proprietary non-fluorescent dye used in the kit becomes strongly fluorescent upon reacting with thiol. The kit can detect as little as 1 picomole of cysteine or GSH in a 100 µL assay volume (10 nM). The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. The thiol sensor used in the kit generates a strongly fluorescent ad-duct upon reacting with a thiol compound. The resulted adduct has the spectral proterties almost identical to those of fluorescein. In addition, both absorption and emission spectra of the thiol adduct are pH-independent, making this assay kit highly robust. The signal can be easily read by a fluorescence microplate reader at Ex/Em = 490/520 nm.

Amplite™ Fluorimetric Fluorescamine Protein Quantitation Kit *Blue Fluorescence*


Protein quantification is necessary in protein purification, electro-phoresis, cell biology, molecular biology, and other research ap-plications. Biuret, Lowry, BCA and Bradford assays are routinely used for estimating protein concentration. However, these colorimetric assays are less sensitive, and require large sample volume to ensure higher accuracy. Our fluorescamine-based protein quantification kit is significantly more sensitive than existing standard colorimetric measurements, e.g., Bradford and Bicinchoninic acid (BCA) assays.

Fluorescamine is intrinsically nonfluorescent but reacts rapidly with primary aliphatic amines, including those in peptides and proteins, to yield a blue-green-fluorescent derivative. The Amplite™ Fluor-escamine Protein Quantitation Kit provides a simple method for quantifying protein concentration in solutions. As little as 3 µg/mL of BSA can be detected. The kit can be performed in a convenient 96-well or 384-well microtiter-plate format. It can be completed within 30 minutes with the fluorescence signal easily monitored at Ex/Em = 380/470 nm. This kit has been used for (1). studying protein/protein interactions; (2). measuring column fractions after affinity chromatography; (3). estimating percent recovery of mem-brane proteins from cell extract; and (4). high-throughput screening of fusion protein.

Related Products


820 25 mg Fluorescamine *UltraPure Grade*




821 5 mg NBD-F [4-Fluoro-7-nitrobenzofurazan] *UltraPure Grade*

825 25 mg NBD-Cl [4-Chloro-7-nitrobenzofurazan] *UltraPure Grade*

21508 5 mg ThioliteTM Green

Figure 2.6. BSA dose response was measured on a 96-well back solid plate with the Amplite™ Fluoremetric Fluorescamine Protein Quantitation Kit. As low as 3 µg/mL of BSA can be detected with 5 minutes incubation time (n=3).

Figure 2.7. GSH and cysteine dose response was measured on a 96-well black solid plate with Am-plite™ Fluorimetric Thiol Quantitation Assay Kit using a NOVOstar microplate reader (BMG Labtech). As low as 10 nM (1 pmol/well) of GSH or Cysteine can be detected with 10 minutes incubation time (n=3).

168737j1_cat.indd 10 2/4/11 11:51 AM



Protein Labeling with Fluorescent D

yes

Protein Labeling with Fluorescent Dyes 3

168737j1_cat.indd 11 2/4/11 11:51 AM



Prot

ein

Labe

ling

with

Flu

ores

cent

Dye

s

3

fluorescent protein labeling kits at-a-glance*

Fluorescent Tag Microscale (100 µg/Reaction)

Production Scale(1 mg/Reaction)

Bulk Scale(10 mg/Reaction)

iFluor™ 488 1224 1225 1226

iFluor™ 555 1227 1228 1229

iFluor™ 594 1230 1231 1232

iFluor™ 647 1235 1236 1237

iFluor™ 680 1240 1241 1242

iFluor™ 700 1245 1246 1247

iFluor™ 750 1250 1251 1252

iFluor™ 780 1255 1256 1257

mFluor™ Violet 420 1105 1106 1107

mFluor™ Violet 450 1100 1101 1102

mFluor™ Violet 560 1114 1115 1116

* products listed by catalog number

168737j1_cat.indd 12 2/4/11 11:51 AM



Related Products

Cat # Size Product Name Alternative to

1023 1 mg iFluor™ 488 succinimidyl ester FITC, Alexa Fluor® 488, DyLight® 488

1028 1 mg iFluor™ 555 succinimidyl ester Cy3®, Alexa Fluor® 555, DyLight® 555

1029 1 mg iFluor™ 594 succinimidyl ester Texas Red®, Alexa Fluor® 594, DyLight® 594


1035 1 mg iFluor™ 680 succinimidyl ester Cy5.5®, Alexa Fluor® 680, DyLight® 680

1036 1 mg iFluor™ 700 succinimidyl ester IRDye® 700, Alexa Fluor® 700


3

iFluor TM 488/555 Labeling

ReadiLink™ iFluor™ 488 Protein Labeling Kits 1

Cat # Size Storage Condition

1224 1 kit (100 µg) < - 15 oC

1225 1 kit (1 mg) < - 15 oC

1226 1 kit (10 mg) < - 15 oC

Cat #1224 (micro scale) is optimized for labeling 100 µg antibody per reactionCat #1225 (production scale) is optimized for labeling 1 mg antibody per reactionCat #1226 (bulk scale) is optimized for labeling 10 mg antibody per reaction.

ReadiLink™ labeling kits require minimal hands-on time without column purification. The kits provide all the essential components with only a simple mixing and spinning step. AAT Bioquest offers three different sizes to meet both exploration research and produc-tion needs.

AAT Bioquest’s iFluor™ dyes are developed for labeling nucleic acids and proteins, in particular, antibodies. These dyes are optimized to have minimal fluorescence quenching effect on proteins and nucleic acids. Our iFluor™ 488 dyes have fluorescence excitation and emission maxima close to 488 nm and 520 nm respectively with good photostability. These spectral characteristics make them excel-lent alternatives to Alexa Fluor® 488 and FITC labeling dyes (Alexa Fluor® is the trademark of Invitrogen). The iFluor™ 488 dye used in the kit is reasonably stable and shows good reactivity and selectiv-ity with proteins. In contrast with pH-dependent FITC fluorescence, both absorption and fluorescence of iFluor™ 488 dyes are indepen-dent to pH change from pH 3 to 11 ReadiLink™ iFluor™ 488 Protein Labeling Kits provide a convenient method to label monoclonal, polyclonal antibodies or other proteins (>10 kDa).



1227 1 kit (100 µg) < - 15 oC

1228 1 kit (1 mg) < - 15 oC

1229 1 kit (10 mg) < - 15 oC


AAT Bioquest’s iFluor™ dyes are developed for labeling nucleic acids and proteins, in particular, antibodies. These dyes are optimized to have minimal fluorescence quenching effect on proteins and nucleic acids. iFluor™ 555 dyes have fluorescence excitation and emission maxima close to 555 nm and 570 nm respectively. These spectral characteristics make them excellent alternatives to Cy3® and Alexa Fluor® 555 (Cy3® and Alexa Fluor® are the trademarks of GE Healthcare and Invitrogen respectively). The iFluor™ 555 dye used in the kit is reasonably stable and shows good reactivity and selectivity with protein amino groups.

These labeling kits have all the essential components with an optimized labeling protocol. Kit 1227 has the labeling dye packed in 5 vials to perform five separate labeling reactions. Each of the five vials of iFluor™ 555 dye provided in the kit is optimized for label-ing ~100 µg antibody. Kit 1227 provides a convenient method to label small amounts of monoclonal, polyclonal antibodies or other proteins (>10 kDa) with the iFluor™ 555 dye. Kits 1228 and 1229 are designed for the protein conjugate productions of medium size and large size respectively.

Figure 3.1. The excitation and emission spectra of iFluor™ 488-Goat Anti-Rabbit IgG conjugate. Figure 3.2. The excitation and emission spectra of iFluor™ 555-Goat Anti-Rabbit IgG conjugate.

www.aatbio.com Protein Labeling with Fluorescent Dyes

168737j1_cat.indd 13 2/4/11 11:51 AM



iFlu

orTM

594

/647

Lab

elin

g

3



1230 1 kit (100 µg) < - 15 oC

1231 1 kit (1 mg) < - 15 oC

1232 1 kit (10 mg) < - 15 oC




1235 1 kit (100 µg) < - 15 oC

1236 1 kit (1 mg) < - 15 oC

1237 1 kit (10 mg) < - 15 oC


iFluor™ 647 dyes have fluorescence excitation and emission maxima close to 647 nm and 660 nm respectively. These spectral charac-teristics make them excellent alternatives to Cy5® and Alexa Fluor® 647 (Cy5® and Alexa Fluor® are the trademarks of GE Healthcare and Invitrogen respectively). iFluor™ 647 dye used in the kit is reason-ably stable and shows good reactivity and selectivity with protein amino groups. The iFluor™ 647 dye has also been used in microar-ray applications in combination with iFluor™ 555. It demonstrated much higher photostability.

These labeling kits have all the essential components with opti-mized labeling protocols. Kit 1235 has the labeling dye packed in 5 vials to perform five separate labeling reactions. Each of the five vials of iFluor™ 647 dye provided in the kit is optimized for label-ing ~100 µg antibody. Kit1235 provides a convenient method to label small amounts of monoclonal, polyclonal antibodies or other proteins (>10 kDa) with the iFluor™ 647 dye. Kits 1236 and 1237 are designed for the protein conjugate productions of medium size and large size respectively.

ReadiLink™ labeling kits require minimal hands-on time without col-umn purification. The kits provide all the essential components for a simple mixing and spinning step. AAT Bioquest offers three different sizes to meet both research and production needs.

AAT Bioquest’s iFluor™ dyes are developed for labeling nucleic acids and proteins, in particular, antibodies. These dyes are optimized to have minimal fluorescence quenching effect on proteins and nucleic acids. Our iFluor™ 594 dyes have fluorescence excitation and emission maxima close to 594 nm and 610 nm respectively with good photostability. Both excitation and fluorescence spectra of iFluor™ 594 dyes are pH-independent in the physiological pH range.These spectral characteristics make them excellent alternatives to Alexa Fluor® 594 and Texas Red® labeling dyes (Alexa Fluor® and Texas Red® are the trademarks of Invitrogen). The iFluor™ 594 dye used in the kit is reasonably stable and shows good reactivity and selectivity with proteins. ReadiLink™ iFluor™ 594 Protein Labeling Kits provide a convenient method to label monoclonal, polyclonal antibodies or other proteins (>10 kDa).

Related Products










Protein Labeling with Fluorescent Dyes www.aatbio.com

168737j1_cat.indd 14 2/4/11 11:51 AM




3

iFluor TM 680/700 Labeling



1240 1 kit (100 µg) < - 15 oC

1241 1 kit (1 mg) < - 15 oC

1242 1 kit (10 mg) < - 15 oC


AAT Bioquest’s iFluor™ dyes are developed for labeling nucleic acids and proteins, in particular, antibodies. These dyes are optimized to have minimal fluorescence quenching effect on proteins and nucleic acids. ReadiLink™ labeling kits require minimal hands-on time without column purification. The kits provide all the essential components with a simple mixing and spinning step. AAT Bioquest offers three different sizes to meet both exploration research and production needs.

Our iFluor™ 680 dyes have fluorescence excitation and emis-sion maxima close to 680 nm and 700 nm respectively with good photostability. These spectral characteristics make them excellent alternatives to Cy5.5®, IRDye® 700 and Alexa Fluor® 680 (Cy5.5®, IRDye® and Alexa Fluor® are the trademarks of GE Healthcare, Li-COR and Invitrogen respectively). The iFluor™ 680 dye used in the kit is reasonably stable and shows good reactivity and selectivity with proteins. ReadiLink™ iFluor™ 680 Protein Labeling Kits provide a convenient method to label monoclonal, polyclonal antibodies or other proteins (>10 kDa). In addition, iFluor™ 680 dye demonstrates good energy transfer efficiency when conjugated to APC.



1245 1 kit (100 µg) < - 15 oC

1246 1 kit (1 mg) < - 15 oC

1247 1 kit (10 mg) < - 15 oC

Cat # 1245 (micro scale) is optimized for labeling 100 µg antibody per reactionCat # 1246 (production scale) is optimized for labeling 1 mg antibody per reactionCat # 1247 (bulk scale) is optimized for labeling 10 mg antibody per reaction.

ReadiLink™ labeling kits require minimal hands-on time without column purification. The kits provide all the essential components with a simple mixing and spinning step. AAT Bioquest offers three different sizes to meet both exploration and production needs. iFluor™ 700 dyes have fluorescence excitation and emission maxima close to 690 nm and 710 nm respectively. These spectral character-istics make them an excellent alternative to Alexa Fluor® 700 (Alexa Fluor® is the trademark of Invitrogen). iFluor™ 700 dye used in the kit is reasonably stable and shows good reactivity and selectivity with protein amino groups.

These labeling kits have all the essential components with opti-mized labeling protocols. Kit 1245 has the labeling dye packed in 5 vials to perform five separate labeling reactions. Each of the five vials of iFluor™ 700 dye provided in the kit is optimized for label-ing ~100 µg antibody. Kit 1245 provides a convenient method to label small amounts of monoclonal, polyclonal antibodies or other proteins (>10 kDa) with the iFluor™ 700 dye. Kits 1246 and 1247 are designed for the protein conjugate productions of medium size and large size respectively.

Related Products








1037 1 mg iFluor™ 750 succinimidyl ester Cy7®, Alexa 9Fluor® 750, DyLight® 750


168737j1_cat.indd 15 2/4/11 11:51 AM




3

iFlu

orTM

750

/780

Lab

elin

g



1250 1 kit (100 µg) < - 15 oC

1251 1 kit (1 mg) < - 15 oC

1252 1 kit (10 mg) < - 15 oC


AAT Bioquest’s iFluor™ dyes are developed for labeling nucleic acids and proteins, in particular, antibodies. These dyes are optimized to have minimal fluorescence quenching effect on proteins and nucleic acids. ReadiLink™ labeling kits require minimal hands-on time without column purification. The kits provide all the essential components with a simple mixing and spinning step. AAT Bioquest offers three different sizes to meet both research and production needs.

Our iFluor™ 750 dyes have fluorescence excitation and emission maxima close to 750 nm and 780 nm respectively with good photo-stability. Our in-house comparable studies indicated that our iFluor 750 dyes are significantly brighter than the corresponding Cy7® and Alexa Fluor® 750 dyes (Cy7® and Alexa Fluor® are the trademarks of GE Healthcare and Invitrogen respectively). These spectral charac-teristics make them superior alternatives to Cy7® and Alexa Fluor® 750. The iFluor™ 750 dye used in the kit is reasonably stable and shows good reactivity and selectivity with proteins. ReadiLink™ iFluor™ 750 Protein Labeling Kit provides a convenient method to label monoclonal, polyclonal antibodies or other proteins (>10 kDa).



1255 1 kit (100 µg) < - 15 oC

1256 1 kit (1 mg) < - 15 oC

1257 1 kit (10 mg) < - 15 oC


iFluor™ 780 dyes have fluorescence excitation and emission maxima close to 780 nm and 800 nm respectively. These spectral characteris-tics make them an excellent alternative to IRDye® 800 (IRDye® is the trademark of Li-COR). iFluor™ 780 dye used in the kit is reasonably stable and shows good reactivity and selectivity with protein amino groups. Our in-house comparable studies indicated that our iFluor™ 780 dyes are significantly brighter than the corresponding IRDye® 800 dyes. This IR dye and its bioconjugates have been extensively used for the in vivo imaging of small animals.

These labeling kits have all the essential components with an optimized labeling protocol. Kit 1255 has the labeling dye packed in 5 vials to perform five separate labeling reactions. Each of the five vials of iFluor™ 780 dye provided in the kit is optimized for label-ing ~100 µg antibody. Kit 1255 provides a convenient method to label small amounts of monoclonal, polyclonal antibodies or other proteins (>10 kDa) with the iFluor™ 780 dye. This kit only requires a simple mix and spin step. Kits 1256 and 1257 are designed for the protein conjugate productions of medium size and large size respectively.

Related Products










168737j1_cat.indd 16 2/4/11 11:52 AM




3

mFluor TM Violet 420/450 Labeling

ReadiLink™ mFluor™ Violet 450 Protein Labeling Kits 1


1100 1 kit (100 µg) < - 15 oC

1101 1 kit (1 mg) < - 15 oC

1102 1 kit (10 mg) < - 15 oC

Cat. #1100 (micro scale) is optimized for labeling 100 µg antibody per reactionCat. #1101 (production scale) is optimized for Labeling 1 mg Antibody Per ReactionCat. #1102 (bulk scale) is optimized for labeling 10 mg protein per reaction.



1105 1 kit (100 µg) < - 15 oC

1106 1 kit (1 mg) < - 15 oC

1107 1 kit (10 mg) < - 15 oC

Cat. #1105 (micro scale) is optimized for labeling 100 µg antibody per reactionCat. #1106 (production scale) is optimized for Labeling 1 mg Antibody Per ReactionCat. #1107 (bulk scale) is optimized for labeling 10 mg protein per reaction.

Related Products









AAT Bioquest’s mFluor™ dyes are developed for flow cytometry-focused applications. These dyes have large Stokes Shifts and can be well excited by the laser lines of flow cytometers (e.g., 405 nm, 488 nm and 633 nm). ReadiLink™ labeling kits require minimal hands-on time without column purification. The kits provide all the essential components with a simple mixing and spinning step. AAT Bioquest offers three different sizes to meet both exploration research and production needs.

Our mFluor™ Violet 420 dyes have fluorescence excitation and emis-sion maxima of ~405 nm and ~420 nm respectively. These spectral characteristics make them another color for the flow cytometric ap-plications using Violet Laser. The mFluor™ Violet 420 SE used in the kit is reasonably stable and shows good reactivity and selectivity with protein amino groups. ReadiLink™ mFluor™ Violet 420 Protein Labeling Kit provides a convenient method to label monoclonal, polyclonal antibodies or other proteins (>10 kDa). It can be also used for labeling other biological molecules.

mFluor™ Violet 450 dyes have fluorescence excitation and emission maxima of ~405 nm and ~450 nm respectively. These spectral char-acteristics make them an excellent alternative to Pacific Orange™ labeling dyes (Pacific Blue™ is the trademark of Invitrogen). The mFluor™ Violet 450 SE used in the kit is reasonably stable and shows good reactivity and selectivity with protein amino groups.

This ReadiLink™ labeling kit has been developed with Goat Anti-Rabbit IgG. It has all the essential components with an optimized labeling protocol for labeling antiboies. Kit 1100 has the labeling dye packed in 5 vials to perform five separate labeling reactions. Each of the five vials of mFluor™ Violet 450 SE provided in the kit is optimized for labeling ~100 µg antibody. Kit 1100 provides a con-venient method to label small amounts of monoclonal, polyclonal antibodies or other proteins (>10 kDa) with the Violet Laser-excit-able mFluor™ Violet 450 SE. Kits 1101 and 1102 are designed for the protein conjugate productions of medium size and large size respectively.

Figure 3.9. The excitation and emission spectra of mFluor™ Violet 420-Goat Anti-Rabbit IgG conjugate. Figure 3.10. The excitation and emission spectra of mFluor™ Violet 450-Goat Anti-Rabbit IgG conjugate.

168737j1_cat.indd 17 2/4/11 11:52 AM




3

mFl

uorTM

Vio

let 5

20/5

60 L

abel

ing



1110 1 kit (100 µg) < - 15 oC

1111 1 kit (1 mg) < - 15 oC

1112 1 kit (10 mg) < - 15 oC


AAT Bioquest’s mFluor™ dyes are developed for flow cytometry-focused applications. These dyes have large Stokes Shifts and can be well excited by the laser lines of flow cytometers (e.g., 405 nm, 488 nm and 633 nm). ReadiLink™ labeling kits require minimal hands-on time without column purification. The kits provide all the essential components with a simple mixing and spinning step. AAT Bioquest offers three different sizes to meet both exploration research and production needs.

Our mFluor™ Violet 520 dyes have fluorescence excitation and emission maxima of ~405 nm and ~520 nm respectively. These spectral characteristics make them another additional color for the flow cytometric applications using Violet Laser. The mFluor™ Violet 520 SE used in the kit is reasonably stable and shows good reactiv-ity and selectivity with protein amino groups. ReadiLink™ mFluor™ Violet 520 Protein Labeling Kit provide a convenient methods to label monoclonal, polyclonal antibodies or other proteins (>10 kDa). It can also be used for labeling other biological molecules.



1114 1 kit (100 µg) < - 15 oC

1115 1 kit (1 mg) < - 15 oC

1116 1 kit (10 mg) < - 15 oC


mFluor™ Violet 560 dyes have fluorescence excitation and emission maxima of ~405 nm and ~560 nm respectively. These spectral char-acteristics make them an excellent alternative to Pacific Orange™ labeling dyes (Pacific Orange™ is the trademark of Invitrogen). The mFluor™ Violet 560 SE used in the kit is reasonably stable and shows good reactivity and selectivity with protein amino groups.

This ReadiLink™ labeling kit has been developed with Goat Anti-Rabbit IgG. It has all the essential components with an optimized labeling protocol for labeling antiboies. Kit 1114 has the labeling dye packed in 5 vials to perform five separate labeling reactions. Each of the five vials of mFluor™ Violet 560 SE provided in the kit is optimized for labeling ~100 µg antibody. Kit 1114 provides a con-venient method to label small amounts of monoclonal, polyclonal antibodies or other proteins (>10 kDa) with the Violet Laser-excit-able mFluor™ Violet 560 SE. Kits 1115 and 1116 are designed for the protein conjugate productions of medium size and large size respectively.

Related Products









Figure 3.11. The excitation and emission spectra of mFluor™ Violet 520-Goat Anti-Rabbit IgG conjugate.

Figure 3.12. The excitation and emission spectra of mFluor™ Violet 560-Goat Anti-Rabbit IgG conjugate.

168737j1_cat.indd 18 2/4/11 11:52 AM


Quantification of Biochem

ical Molecules

Quantification of Biochemical Molecules 4

168737j1_cat.indd 19 2/4/11 11:52 AM



Qua

ntifi

catio

n of

Bio

chem

ical

Mol

ecul

es

4

biochemical assay kits at-a-glance*

Biochemical Assay Absorption Fluorescence Luminescence

Acetylcholine Assay 11403

ADP Assay 21655

Aldehyde Quantitation 10051 10052

ATP Assay 21609 & 21610

Calcium Quantitation 36360

Cholesterol Quantitation 40006

Choline Quantitation 40007

Ethanol Quantitation 40001

Glucose Quantitation 40005

Glutamic Acid Assay 10054

Glutathione Assay 10055

Glutathione GSH/GSSG Ration Assay 10056

Hydrogen Peroxide Assay 11501 & 11502

NAD/NADH Assay 15258 15257

NAD/NADH Ratio Assay 15263

NADH Assay 15261

NADH/NADPH Assay 15260 15259

NADH/NADPH Ratio Assay 15264

NADPH Assay 15262

Phosphate Assay 21659 & 21665 21660

Pyrophosphate Assay 21611


168737j1_cat.indd 20 2/4/11 11:52 AM



www.aatbio.com Quantification of Biochemical MoleculesAcetylcholine/A

DP

PhosphoWorks™ Fluorimetric ADP Assay Kit *Red Fluorescence*


All signal transduction pathways are regulated on some level by phosphorylation, making phosphorylation relevant to most, if not all, areas of cell signaling and neuroscience research. Kinases are of interest to researchers involved in drug discovery due to their broad relevance to diseases. Most of the commercial protein kinase assay kits are either based on monitoring the phosphopeptide formation or ATP depletion. For the kinase assay kits that are based on the detection of phosphopeptides, one has to spend time and efforts to identify an optimized peptide substrate.

The PhosphoWorks™ Fluorimetric ADP Assay Kit is used for monitor-ing ADP formation, which is directly proportional to enzyme phos-photransferase activity and is measured fluorimetricaly. This kit pro-vides a fast, simple, and homogeneous assay for the measurement of kinase activities. It is a non-radioactive and no wash method to detect the amount of ADP produced as a result of enzyme activity. Its characteristics of high sensitivity (<0.3 μM ADP) and broad ATP tolerance (1-300 μM) make it an ideal kit for determining kinase Michaelis-Menten kinetics as well as for screening and identifying kinase inhibitors. The kit has been used for screening kinase inhibi-tors and profiling kinase activities.

4

Amplite™ Fluorimetric Acetylcholine Assay Kit *Red Fluorescence*


Acetylcholine (ACh) and its metabolites are involved in three main physiological purposes: structural integrity and signaling roles for cell membranes, cholinergic neurotransmission (acetylcholine syn-thesis), and as a major source for methyl groups via its metabolite. Acetylcholine is a neurotransmitter in both the central and periph-eral nervous systems. It is one of many neurotransmitters in the au-tonomic nervous system (ANS) and the only neurotransmitter used in the motor division of the somatic nervous system. It is involved in a number of biological events that are related to diabetic vasculopa-thy, hypertension, and Alzheimer’s disease.

Our Amplite™ Fluorimetric Acetylholine Assay Kit provides one of the most sensitive methods for quantifying acetylcholine. The kit uses Amplite™ Red to quantify acetylcholine through the choline oxidase-mediated enzyme coupling reactions. The fluorescence in-tensity of Amplite™ Red is proportional to acetylcholine formation. The kit is an optimized “mix and read” assay. It provides an ultrasen-sitive one-step fluorimetric assay to detect as little as 0.01 nano-moles ACh in a 100 µL assay volume (0.1μM). Its signal can be easily read with a fluorescence microplate reader at Ex/Em = ~540/590 nm or an absorbance microplate reader at ~576 nm.

Related Products


11000 25 mg Amplite™ ADHP [10-Acetyl-3,7-dihydroxyphenoxazine]

11400 1 mg Amplite™ Colorimetric Acetylcholinesterase Assay Kit

11401 1 kit Amplite™ Fluorimetric Acetylcholinesterase Assay Kit *Green Fluorescence*

11502 1 kit Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit *Near Infrared Fluorescence*

11501 1 kit Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit *Red Fluorescence*

11009 1 kit Amplite™ IR

21611 1 kit PhosphoWorks™ Fluorimetric Pyrophosphate Assay Kit *Blue Fluorescence*

Figure 4.2. ADP dose response was measured with the PhosphoWorks™ Fluorimetric ADP Assay Kit in a 384-well black solid plate. As low as 0.3 µM of ADP can be detected in 30 minutes incubation time .

Figure 4.1. Acetylcholine dose response was measured in a 96-well black solid plate with Amplite™ Fluorimetric Acetylcholine Assay Kit (Cat. # 11403) using a Gemini fluorescence microplate reader (Molecular devices). As low as 0.01 nmoles/well (0.1µM) of acetylcholine can be detected with 10 minutes incubation time (n=3).

168737j1_cat.indd 21 2/4/11 11:52 AM



Quantification of Biochemical Molecules www.aatbio.com

Related Products


15258 1 kit Amplite™ Colorimetric NAD/NADH Assay Kit

15260 1 kit Amplite™ Colorimetric NADP/NADPH Assay Kit

40001 1 kit Amplite™ Ethanol Quantitation Kit

15255 1 kit Amplite™ Fluorimetric Lysyl Oxidase Assay Kit *Red Fluorescence*

15257 1 kit Amplite™ Fluorimetric NAD/NADH Assay Kit *Red Fluorescence*

11303 1 kit Amplite™ Monoamine Oxidase Assay Kit *Red Fluorescence*

15265 1 kit ReadiUse™ NADPH Regenerating Kit

4

Ald

ehyd

e Q

uant

itatio

n

Amplite™ Fluorimetric Aldehyde Quantitation KitCat # Size Storage Condition

10052 1 kit < - 15 oC

The formation, reactivity and toxicity of aldehydes originating from the peroxidation of lipids of cellular membranes have received great attention in recent years. Rapid and accurate measurement of aldehydes is an important task for biological research, chemical research, food industry and environmental pollution surveillance. There are a few reagents or assay kits available for quantifying the number of aldehydes. Most of the existing aldehyde test methods are based on separations either by the tedious and expensive HPLC-MS or GC-MS.

Both our Amplite™ Colorimetric Aldehyde Qutitation Kit (10051) and Amplite™ Fluorimetric Aldehyde Qutitation kit (10052) are used for quantifying aldehydes at higher pH. Kit 10052 uses a proprietary fluorogenic dye that generates a strongly fluorescent product upon reacting with an aldehyde. Kit 10052 is much more sensitive than Kit 10051. This fluorimetric kit provides a sensitive mix-and-read method to detect as little as 0.1 nanomole of aldehyde in a 100 µL assay volume (1 µM). The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. Its signal can be easily read by a fluorescence microplate reader at Ex/Em = 365/435 nm.

Amplite™ Colorimetric Aldehyde Quantitation KitCat # Size Storage Condition

10051 1 kit < - 15 oC

Very reactive aldehydes, namely 4-hydroxyalkenals, were first shown to be formed in autoxidizing chemical systems. It was subsequently shown that 4-hydroxyalkenals, particularly 4-hydroxynonenal, were formed in substantial amounts under biological conditions, i.e. during the peroxidation of lipids of liver microsomes incubated in the NADPH-Fe system. Many other aldehydes were also identified in peroxidizing liver microsomes or hepatocytes, e.g., alkanals , alk-2-enals, and 4-hydroxyalkenals.

Most of the existing aldehyde test methods are based on separa-tions either by the tedious and expensive HPLC-MS or GC-MS. Our Amplite™ Colorimetric Aldehyde Quantitation kit uses a proprietary dye that generates a chromogenic product upon reacting with an aldehyde. The kit provides a sensitive, one-step colorimetric method to detect as little as 1 nanomole of aldehyde in a 100 µL assay vol-ume (10 µM). The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and readily adapted to automation without a separation step. Its signal can be easily read with an ab-sorbance microplate reader at 405 or 550 nm. This kit has been used for monitoring activities of oxidases that convert an amino group to an aldehyde group.

Figure 4.3. Aldehyde dose response was measured in a 96-well clear plate with Amplite™ Colorimetric Aldehyde Quantitation Assay Kit. As low as 10 µM (1 nanomol/well) of aldehyde can be detected.

Figure 4.4. Formaldehyde dose response was measured in a 96-well black plate with Amplite™ Fluorimetric Aldehyde Quantitation Kit. As low as 1 µM (0.1 nanomol/well) of formaldehyde can be detected with 15 minutes incubation time (n=3).

168737j1_cat.indd 22 2/4/11 11:52 AM



www.aatbio.com Quantification of Biochemical Molecules

4

ATP

PhosphoWorks™ Luminometric ATP Assay Kit*Steady Glow*


Firefly luciferase is a monomeric 61 kD enzyme that catalyses a two-step oxidation of luciferin, which yields light at 560 nm. The first step involves the activation of the protein by ATP to produce a reactive mixed anhydride intermediate. In the second step, the ac-tive intermediate reacts with oxygen to create a transient dioxetane, which quickly breaks down to the oxidized product oxyluciferin and carbon dioxide along with a burst of light. When ATP is the limiting component, the intensity of light is proportional to the concentra-tion of ATP. Thus the measurement of the light intensity can be used to quantify ATP with a luminometer.

As Kit 21610, this assay is also based on the detection of ATP using firefly luciferase to catalyze the release of light with ATP and lucif-erin. Complementary to Kit 21610, this kit provides a more stable luminescence signal that lasts for a few hours, making it convenient to be used with the luminometers that are not equipped with liquid handling capacity. Although Kit 21609 is less sensitive than Kit 21610, its more stable signal provides advantages for some particular applications, such as rapid diagnostic applications. This kit has been used successfully for rapid food safety inspection with a hand-held luminometer.

PhosphoWorks™ Luminometric ATP Assay Kit *Bright Glow*


Adenosine triphosphate (ATP) plays a fundamental role in cel-lular energenics, metabolic regulation and cellular signaling. The quantitation of ATP can be used for a variety of biological applica-tions. Because ATP is the energy source for almost all living organ-isms and rapidly degrades in the absence of viable organisms, its existence can be used to identify the presence of viable organisms. The measurement of ATP has been used for cell cytoxicity, detection of bacteria on surfaces, quantification of bacteria in water, somatic cells in culture and food quality.

The PhosphoWorks™ Luminometric ATP Assay Kit comes with all the essential components and provides a fast, simple and homoge-neous luminescence assay for the determination of cell proliferation and cytotoxicity in mammalian cells. This assay is based on the de-tection of ATP using firefly luciferase to catalyze the release of light by ATP and luciferin. It can be performed in a convenient 96-well or 384-well microtiter-plate format and run with many luminescence instruments. The assay is extremely sensitive and can detect 10-100 cells/well. Its high sensitivity permits the detection of ATP in many biological systems, environmental samples and foods. This assay kit has higher sensitivity while Kit 21609 has more stable signal.

Figure 4.5. CHO-K1 cell number was measured with the PhosphoWorks™ Luminescence ATP Assay Kit on a 96-well white plate using a NOVOstar plate reader (BMG Labtech). The kit can detect as low as 10 cells per well. The integration time was 1 sec.

Figure 4.6. ATP dose response was measured with the PhosphoWorks™ Luminescence ATP Assay Kit ATP concentrations from 10 μM to 0.1 nM was monitored for up to 5 hours (above figure shows 20 minutes, 1, 2, 3, 4, and 5 hours signal).

Related Products


12500 1 mL Luciferase *Recombinant firefly*

12512 1 mg D-Luciferin phosphate

12507 1 g D-Luciferin, potassium salt *UltraPure Grade*

21655 1 kit PhosphoWorks™ Fluorimetric ADP Assay Kit *Red Fluorescence*

21660 1 kit PhosphoWorks™ Fluorimetric Phosphate Assay Kit *Red Fluorescence*


11629 5 mg SunRed™ Phosphate

168737j1_cat.indd 23 2/4/11 11:52 AM




4

Cal

cium

/Cho

lest

erol

Related Products


40007 1 kit Amplite™ Choline Quantitation Kit

10051 1 kit Amplite™ Colorimetric Aldehyde Quantitation Kit

11403 1 kit Amplite™ Fluorimetric Acetylcholine Assay Kit *Red Fluorescence*

10052 1 kit Amplite™ Fluorimetric Aldehyde Quantitation Kit

40001 1 kit Amplite™ Fluorimetric Ethanol Quantitation Kit

40005 1 kit Amplite™ Fluorimetric Glucose Quantitation Kit *Red Fluorescence*

10054 1 kit Amplite™ Fluorimetric Glutamic Acid Assay Kit *Red Fluorescence*

Amplite™ Cholesterol Quantitation Kit


40006 1 kit < - 15 oC

Cholesterol is required to build and maintain cell membranes. It modulates membrane fluidity over the range of physiological temperatures. Within cells, cholesterol is the precursor molecule in several biochemical pathways. Cholesterol is also an important precursor molecule for the synthesis of Vitamin D and the steroid hormones, including the adrenal gland hormones cortisol and aldosterone as well as the sex hormones progesterone, estrogens, together with testosterone and their derivatives.

This Amplite™ Cholesterol Quantitation Assay Kit provides one of the most sensitive methods for quantifying cholesterol. The kit uses Amplite™ Red to quantify the concentration of cholesterol, which is related to the production of hydrogen peroxide in the cholesterol oxidase-mediated enzyme coupling reactions in the presence of cholesterol. The amount of cholesterol is proportional to the con-centration of hydrogen peroxide formed in the enzyme coupling reaction cycle. In the presence of peroxidase, the fluorescence intensity of Amplite™ Red is proportional to the concentration of hydrogen peroxide that is converted to the concentration of choles-terol. The assay can be readily read with a fluorescence microplate reader at Ex/Em = ~540/590 nm.

Figure 4.8. Cholesterol dose response on 96-well black solid plate using a Gemini (Molecular Devices) measured with Amplite™ Fluorimetric Cholesterol Assay Kit. As low as 0.03 µM cholesterol can be detected with 30 minutes incubation time (n=3).

Amplite™ Calcium Quantitation Kit *Red Fluorescence*


36360 1 kit < - 15 oC

Calcium is essential for all living organisms, particularly in cell physiology, where the movement of calcium ion into and out of the cytoplasm functions as a signal for many cellular processes. Calcium also plays an important role in mediating the constriction and relax-ation of blood vessels, nerve impulse transmission, muscle contrac-tion, and hormone secretion. The serum level of calcium is closely regulated (9 to 10.5 mg/dL) in the human body. Both hypocalcemia and hypercalcemia are serious medical disorders.

Amplite™ Calcium Quantitation Kit provides a simple method for detecting calcium in physiology solutions by using our proprietary red fluorescence probe. The fluorescence signal can be easily read with a fluorescence microplate reader at Ex/Em = 540/590 nm. The kit can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. The assay can be completed within 30 minutes. With the Am-plite™ Calcium Quantitation Kit, we have detected as little as 0.03 mM calcium. The kit has a broad dynamic range (30 µM to 10 mM). If more sensitive calcium detection is required, we recommend that Quest Fluo-8™ or Fluo-3 be used instead. Both Quest Fluo-8™ and Fluo-3 can be used for determining calcium in nM range.

Figure 4.7. Calcium dose response was measured in a 96-well black solid plate with the Amplite™ Calcium Quantitation Kit. As low as 0.03 mM calcium can be detected with 5 minutes incubation time (n=3).

168737j1_cat.indd 24 2/4/11 11:52 AM




Related Products


40006 1 kit Amplite™ Cholesterol Quantitation Kit

10051 1 kit Amplite™ Colorimetric Aldehyde Quantitation Kit



11402 1 kit Amplite™ Fluorimetric Acetylcholinesterase Assay Kit *Red Fluorescence*

10052 1 kit Amplite™ Fluorimetric Aldehyde Quantitation Kit

40005 1 kit Amplite™ Glucose Quantitation Kit

4

Choline/Ethanol

Figure 4.10. Ethanol dose response was measured with Amplite™ Fluorimetric Ethanol Quantitation Kit on a 96-well black solid plate using a Gemini Fluorescence microplate reader (Molecular Devices). As low as 0.0003% of Ethanol was detected with 15 minutes incubation time (n=3).

Figure 4.9. Choline dose response was measured in a 96-well solid black plate with Amplite™ Fluori-metric Choline Assay Kit (40007) using a Gemini fluorescence microplate reader (Molecular Devices). As low as 0.1µM (10 picomoles/well) of choline can be detected with 10 minutes incubation time (n=3).

Amplite™ Choline Quantitation KitCat # Size Storage Condition

40007 1 kit < - 15 oC

Choline and its metabolites play an important role in the structural integrity and signaling of cell membranes and cholinergic neuro-transmission (acetylcholine synthesis). It is a major source for methyl groups via its metabolite, trimethylglycine that participates in the S-adenosylmethionine synthesis pathways. Choline deficiency may cause liver disease, atherosclerosis and possibly neurological disorders. Despite its importance in the central nervous system as a precursor for acetylcholine and membrane phosphatidylcholine, the role of choline in mental illness has been little studied.

This Amplite™ Choline Quantitation Kit provides one of the most sensitive methods for quantifying choline. The kit uses Amplite™ Red to quantify the concentration of choline, which is related to the production of hydrogen peroxide in the choline oxidase-mediated enzyme coupling reactions. The amount of choline is proportional to the concentration of hydrogen peroxide formed in the enzyme coupling reaction cycle. In the presence of peroxidase, the fluores-cence intensity of Amplite™ Red is proportional to the formation of hydrogen peroxide that is converted to the concentration of cho-line. The assay can be readily read with a fluorescence microplate reader at Ex/Em = ~540/590 nm. Alternatively the assay can also be read at ~570 nm with an absorption microplate reader.

Amplite™ Fluorimetric Ethanol Quantitation KitCat # Size Storage Condition

40001 1 kit < - 15 oC

Ethanol is a powerful psychoactive drug and one of the oldest recreational drugs. It is best known as the type of alcohol found in alcoholic beverages and thermometers. In common usage, it is often referred to simply as alcohol or spirits. Ethanol is a central ner-vous system depressant and has significant psychoactive effects in sublethal doses. A blood ethanol level of 0.5% or more is commonly fatal. Ethanol levels of even less than 0.1% can cause intoxication, with unconsciousness often occurring at 0.3–0.4%. The amount of ethanol in the body is typically quantified by blood alcohol content.

The ability to rapidly perform quantitative measurements of ethanol is highly desirable in life science research, clinical evaluations, food, and pharmaceutical industries. Our non-radioactive ethanol assay is based on the oxidation of ethanol by alcohol oxidase. The kit uses our Amplite™ Red reagent that makes the kit recordable in a dual mode, the fluorescent signal can be easily read by a fluorescence microplate reader at Ex/Em = ~540/590 nm, or its absorption can be readily read by an absorbance microplate reader at ~570 nm. The Amplite™ Fluorimetric Ethanol Quantitation Kit can be performed in a convenient 96-well or 384-well microtiter-plate format. The assay can be completed within 30 minutes and as little as 0.0003% ethanol can be detected.

168737j1_cat.indd 25 2/4/11 11:52 AM




Related Products



40006 1 kit Amplite™ Cholesterol Quantitation Kit


40001 1 kit Amplite™ Fluorimetric Ethanol Quantitation Kit



11009 1 kit Amplite™ IR

4

Glu

cose

/Glu

tam

ic A

cid

Amplite™ Fluorimetric Glucose Quantitation Kit*Red Fluorescence*


40005 1 kit < - 15 oC

Glucose, a monosaccharide, is the most important carbohydrate in biology. It is a source of energy and metabolic intermediate for cell growth. As one of the main products of photosynthesis, glucose starts cellular respiration in both prokaryotes and eukaryotes. Glucose level is a key diagnostic parameter for many metabolic disorders, e.g., diabetes.

This Amplite™ glucose assay kit provides a quick and sensitive method for the measurement of glucose. It uses glucose oxidase-based enzyme coupled reactions to detect glucose through the pro-duction of hydrogen peroxide, which is monitored by our Amplite™ Red peroxidase substrate. Amplite™ Red peroxidase substrate can be recorded in a dual mode, the fluorescent signal can be easily read by a fluorescence microplate reader at Ex/Em = 540/590 nm, or its absorption can be read by an absorbance microplate reader at ~570 nm. The assay is robust, and can be readily adapted for a wide variety of applications that require the measurement of glucose. The assay has very low background since it is run in the red visible range that significantly reduces the interference from biological samples. It has demonstrated high sensitivity and low interference with excitation at 570 nm and emission at 590 nm. With the Amplite™ Glucose Quantitation Kit, we can detect as little as 3 μM D-glucose.

Figure 4.11. Glucose dose response was measured with Amplite™ Fluorimetric Glucose Quantitation Kit on a 96-well black solid plate using a Novostar microplate reader (BMG Labtech). As low as 3 µM glucose was detected with 30 minutes incubation time (n=3).

Amplite™ Fluorimetric Glutamic Acid Assay Kit *Red Fluorescence*


10054 1 kit < - 15 oC

Glutamate is a key molecule in cellular metabolism. It is the most abundant excitatory neurotransmitter in the vertebrate nervous system. Nerve impulses trigger release of glutamate from the pre-synaptic cells. In the opposing post-synaptic cell, glutamate recep-tors, such as the NMDA receptor, bind glutamate and are activated. Glutamate works not only as a point-to-point transmitter but also as a spill-over synaptic crosstalk between synapses.

This Amplite™ Fluorimetric Glutamic Acid Assay Kit provides one of the most sensitive methods for quantifying glutamic acid. The kit uses Amplite™ Red to quantify the concentration of glutamic acid, which is related to the production of hydrogen peroxide in the glu-tamate oxidase-mediated enzyme coupling reactions in the pres-ence of glutamic acid. The amount of glutamic acid is proportional to the concentration of hydrogen peroxide formed in the enzyme coupling reaction cycle. In the presence of peroxidase, the fluores-cence intensity of Amplite™ Red is proportional to the formation of hydrogen peroxide that can be converted to the concentration of glutamic acid. The assay can be readily read with a fluorescence microplate reader at Ex/Em = ~540/590 nm. Alternatively the assay can also be read at ~570 nm with an absorption microplate reader, but the sensitivity is reduced by ~10 times.

Figure 4.12. Glutamic acid dose response was measured with Amplite™ Glutamic Acid Assay Kit in a 96-well black solid plate using a Gemini fluorescence microplate reader (Molecular Devices). As low as 40 nM glutamic acid was detected with 30 minutes incubation time (n=3).

168737j1_cat.indd 26 2/4/11 11:52 AM




Related Products



5525 1 kit Amplite™ Colorimetric Maleimide Quantitation Kit

5523 1 kit Amplite™ Fluorimetric Maleimide Quantitation Kit *Green Fluorescence*

5524 1 kit Amplite™ Fluorimetric Thiol Quantitation Assay Kit *Green Fluorescence*

22810 1 kit Cell Meter™ Intracellular GSH Assay Kit *Optimized for Flow Cytometry*

21507 5 mg Thiolite™ Blue


4

Glutathione

Amplite™ Fluorimetric Glutathione Assay Kit *Green Fluorescence*


10055 1 kit < - 15 oC

The monitoring of reduced and oxidized glutathione (GSH) in bio-logical samples is essential for evaluating the redox and detoxifica-tion status of cells and tissues in relation to the protective role of glutathione against oxidative and free-radical-mediated cell injury. Cysteine metabolism disorders include cystinosis, an autosomal recessive disease produced by a defect in lysosomal transport, and cystinuria, a common heritable disorder of amino acid transport. Cysteine is unique among the amino acids found in proteins.

There are a few reagents or assay kits available for quantitating thiols in biological systems. However, all the commercial kits either lack sensitivity or have tedious protocols. Our Amplite™ Fluorimetric GSH Qutitation Kit provides an ultrasensitive fluorimetric assay to quantitate GSH in sample. The kit uses a proprietary non-fluorescent dye that becomes strongly fluorescent upon reacting with thiol. The kit provides a sensitive, one-step fluorimetric method to detect as little as 1 picomole of cysteine or GSH in a 100 µL assay volume. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. Its signal can be easily read using a fluorescence microplate reader at Ex/Em = 490/520 nm.

Amplite™ Fluorimetric Glutathione GSH/GSSG Ratio Assay Kit *Green Fluorescence*


10056 1 kit < - 15 oC

When cells are exposed to increased levels of oxidative stress, GSSG will accumulate and the ratio of GSH to GSSG will decrease. The glutathione redutase recycles GSSG to GSH with simultaneous oxidation of b-nicotinamide adenine dinuclecotide phosphate. The monitoring of GSH/GSSG ratio and the quantification of GSSG in biological samples are essential for evaluating the redox and detoxi-fication status of cells and tissues in relation to the protective role of glutathione against oxidative and free-radical-mediated cell injury.

There are a few reagents or assay kits available for the quantita-tion of thiols in biological systems. However, all the commercial kits either lack sensitivity or have tedious protocols. Our Amplite™ Fluorimetric GSH/GSGG Ratio Kit provides an ultrasensitive assay to quantitate GSH in the sample. The kit uses a proprietary non-fluorescent dye that becomes strongly fluorescent upon reacting with thiol. The kit provides a sensitive, one-step fluorimetric method to detect as little as 1 picomole of cysteine or GSH in a 100 µL assay volume. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. Its signal can be easily read by a fluores-cence microplate reader at Ex/Em = 490/520 nm.

Figure 4.14. GSH and Total GSH (GSH+GSSG) dose response were measured with Amplite™ Fluorimet-ric Glutathione GSH/GSSG Ratio Assay Kit. Blue line (A), in the presence of GSH only; Red line (B), in the presence of 1:1 GSH/GSSG.

Figure 4.13. GSH dose response was measured in a 96-well black solid plate with Amplite™ Fluori-metric Glutathione Assay Kit using a NOVOStar microplate reader (BMG Labtech). As low as 10 nM (1 pmol/well) of GSH can be detected with 10 minutes incubation time (n=3).




4

Hyd

roge

n Pe

roxi

de

Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit *Near Infrared Fluorescence*


11502 1 kit < - 15 oC

This Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit uses our unique Amplite™ IR peroxidase substrate to quantify hydrogen peroxide in solutions and cell extracts. Amplite™ IR generates the fluorescence that is pH-independent from pH 4 to 10. It is a superior alternative to ADHP (Amplex Red™) for the detections that require low pH where ADHP has reduced fluorescence. In addition, Am-plite™ IR generates a product that has maximum absorption at 647 nm with maximum emission at 670 nm. This near infrared fluores-cence minimizes the assay background that is often caused by the autofluorescence of biological samples. It can also be used to detect a variety of oxidase activities through enzyme-coupled reactions.

This Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit provides a sensitive, one-step fluorometric assay to detect as little as 3 picomoles of H2O2 in a 100 µL assay volume (30 nM). The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. Its signal can be easily read by either a fluorescence microplate reader at Ex/Em = ~640/680 nm or an absorbance microplate reader at ~650 nm. Due to its long emission wavelength, this kit has low interference from biological samples.

Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit *Red Fluorescence*


11501 1 kit < - 15 oC

Hydrogen peroxide (H2O2) is a reactive oxygen metabolic by-prod-uct that serves as a key regulator for a number of oxidative stress-related states. It is involved in a number of biological events that have been linked to asthma, atherosclerosis, diabetic vasculopathy, osteoporosis, neurodegenerative diseases and Down’s syndrome. Measurement of this reactive species will help to determine how oxidative stress modulates a variety of intracellular pathways.

This Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit uses our non-fluorescent Amplite Red™ peroxidase substrate to quantify hydrogen peroxide in solutions and cell extracts. It can also be used to detect a variety of oxidase activities through enzyme-coupled reactions. The kit is an optimized “mix and read” assay that is com-patible with HTS liquid handling instruments. It provides a sensitive, one-step fluorometric assay to detect as little as 3 picomoles of H2O2 in a 100 µL assay volume (30 nM). The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and readily adapted to automation. Its signal can be easily read by either a fluorescence microplate reader at Ex/Em = ~540/590 nm or an absorbance microplate reader at ~570 nm.

Figure 4.16. H2O2 dose response was measured in a 384-well black solid plate with the Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit. As low as 0.03 µM H2O2 can be detected with 30 minutes incubation time (n=3).

Figure 4.15. H2O2 dose response was measured in a 96-well black solid plate with the Amplite™Fluorimetric Hydrogen Peroxide Assay Kit. As low as 0.03 µM H2O2 can be detected.

Related Products



11551 1 kit Amplite™ Colorimetric Peroxidase Assay Kit *Blue Color*

11540 1 kit Amplite™ Fluorimetric Goat Anti-Mouse IgG-HRP Conjugate ELISA Assay Kit *Red Fluorescence*

11541 1 kit Amplite™ Fluorimetric Goat Anti-Rabbit IgG-HRP Conjugate ELISA Assay Kit *Red Fluorescence*

11552 1 kit Amplite™ Fluorimetric Peroxidase Assay Kit *Red Fluorescence*

11009 1 mg Amplite™ IR

11001 1 L ReadiUse™ ABTS Solution *Optimized for ELISA Assays with HRP Conjugates*




Related Products



15261 1 kit Amplite™ Fluorimetric NADH Assay Kit *Red Fluorescence*

15263 1 kit Amplite™ Fluorimetric NAD/NADH Ratio Assay Kit *Red Fluorescence*

15262 1 kit Amplite™ Fluorimetric NADPH Assay Kit *Red Fluorescence*

15259 1 kit Amplite™ Fluorimetric NADP/NADPH Assay Kit *Red Fluorescence*

15264 1 kit Amplite™ Fluorimetric NADP/NADPH Ratio Assay Kit *Red Fluorescence*


4

NA

D/N

AD

H

Amplite™ Colorimetric NAD/NADH Assay Kit


15258 1 kit < - 15 oC

The existing NAD/NADH methods suffer low sensitivity and high interference since the assays are done in the UV range. Our Am-plite™ Colorimetric NAD/NADH Assay Kit provides a convenient method for sensitive detection of NAD and NADH. The enzymes in the system specifically recognize NAD/NADH in an enzyme cycling reaction that significantly increases detection sensitivity. In addi-tion, this assay has very low background since it is performed in the red visible range that considerably reduces the interference resulted from biological samples. There is also no need to purify NAD/NADH from sample mix. The assay has demonstrated high sensitivity and low interference with absorbance at ~576 nm.

The Amplite™ Colorimetric NAD/NADH Assay Kit provides a sensi-tive, one-step assay to detect as little as 30 picomoles of NAD(H) in a 100 µL assay volume (300 nM). The assay can be performed in a convenient 96-well or 384-well microtiter-plate format. Its signal can be easily read by an absorbance microplate reader at ~575 nm or at the absorbance ratio of ~570 nm to ~605 nm to increase assay sensitivity.

Amplite™ Fluorimetric NAD/NADH Assay Kit *Red Fluorescence*


15257 1 kit < - 15 oC

The traditional NAD/NADH and NADP/NADPH assays are run by monitoring the changes in NADH or NADPH absorption at 340 nm. This Amplite™ Fluorimetric NAD/NADH Assay Kit provides a con-venient method for sensitive detection of NAD and NADH. The en-zymes in the system specifically recognize NAD/NADH in an enzyme cycling reaction that significantly increases detection sensitivity. In addition, this assay has very low background since it is run in the red visible range that considerably reduces the interference resulted from biological samples. The assay has demonstrated high sensitiv-ity and low interference at Ex/Em = 540/590 nm.

The Amplite™ Fluorimetric NAD/NADH Assay Kit provides a sensi-tive, one-step assay to detect as little as 10 picomoles of NAD(H) in a 100 µL assay volume (100nM). The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and readily adapted to automation without a separation step. Its signal can be easily read by either a fluorescence microplate reader at Ex/Em = 530-570 nm/590-600 nm (maximum Ex/Em = 540/590 nm) or an absorbance microplate reader at ~576 nm.

Figure 4.18. NADH dose response was measured with Amplite™ Fluorimetric NAD/NADH Assay Kit in a 96-well black solid plate. As low as 100 nM (10 pmol/well) of NADH can be detected with 1 hour incubation time (n=3) while there is no response from NADPH.

Figure 4.17. NADH dose response was measured with Amplite™ Colorimetric NAD/NADH Assay Kit in a 96-well white wall/clear bottom plate using a NOVOStar (BMG Labtech) microplate reader. As low as 300 nM (30 pmol/well) of NADH can be detected with 1 hour incubation time (n=3) while there is no response from NADPH.

168737j1_cat.indd 29 2/4/11 11:52 AM




Related Products









4

NA

D/N

AD

H

Amplite™ Fluorimetric NAD/NADH Ratio Assay Kit *Red Fluorescence*


15263 1 kit < - 15 oC

Nicotinamide adenine dinucleotide (NAD+) and nicotinamide ad-enine dinucleotide phosphate (NADP+) are two important cofactors found in cells. The traditional NAD/NADH and NADP/NADPH assays suffer low sensitivity and high interference since they are done in the UV range.

This Amplite™ Fluorimetric NAD/NADH Ratio Assay Kit provides a convenient method for sensitive detection of NAD, NADH and their ratio. The enzymes in the system specifically recognize NAD/NADH in an enzyme cycling reaction that significantly increases detection sensitivity. In addition, this assay has very low background since it is run in the red visible range that considerably reduces the interfer-ence from biological samples. There is no need to purify NAD/NADH from sample mix. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format. Its signal can be easily read by either a fluorescence microplate reader at Ex/Em = 530-570/590-600 nm (maximum Ex/Em = 540/590 nm) or an absorbance microplate reader at ~576 nm. This kit provides NAD and NADH ex-traction buffer, and cell lysis buffer for your convenience. This kit has been frequently used for determining NAD/NADH from cell lysates.

Figure 4.19. Total NADH and NAD, and their extract dose response were measured with Amplite™ Fluorimetric NAD/NADH Ratio Assay Kit in a 96-well black solid plate. The blank signal was subtracted from the values for those wells with the NADH reactions.

Amplite™ Fluorimetric NADH Assay Kit*Red Fluorescence*


15261 1 kit < - 15 oC

The traditional NAD/NADH and NADP/NADPH assays are done by monitoring the changes in NADH or NADPH absorption at 340 nm. The short UV wavelength of the traditional NAD/NADH and NADP/NADPH assays makes these methods to suffer low sensitivity and high interference. Due to the weak absorption of NAD and NADH, the UV absorption method requires large sample sizes, making the same NAD and NADH measurement unpractical if the availability of samples is limited.

This Amplite™ Fluorimetric NADH Assay Kit provides a convenient method for the detection of NADH. The enzymes in the system specifically recognize NADH in an enzyme recycling reaction. In addition, this assay has very low background since it is run in the red visible range that significantly reduces the interference resulted from biological samples. The Amplite™ Fluorimetric NADH Assay Kit provides a sensitive, one-step assay to detect as little as 100 pico-moles of NADH in a 100 µL assay volume (1 µM). The assay can be performed in a convenient 96-well or 384-well microtiter-plate for-mat. Its signal can be easily read by either a fluorescence microplate reader at Ex/Em = 540/590 nm or an absorbance microplate reader at ~576 nm.

Figure 4.20. NADH dose response was measured with Amplite™ Fluorimetric NADH Assay Kit in a 96-well black solid plate. As low as 1 µM (100 pmol/well) of NADH can be detected with 1 hour incubation time (n=3) while there is no response from NAD.

168737j1_cat.indd 30 2/4/11 11:53 AM




Related Products









4

NA

DP/N

AD

PH

Amplite™ Colorimetric NADP/NADPH Assay Kit


15260 1 kit < - 15 oC

Nicotinamide adenine dinucleotide (NAD+) and nicotinamide ad-enine dinucleotide phosphate (NADP+) are two important cofactors found in cells. This Amplite™ Colorimetric NADP/NADPH Assay Kit provides a convenient method for sensitive detection of NADP and NADPH. The enzymes in the system specifically recognize NADP/NADPH in an enzyme cycling reaction that significantly increases detection sensitivity. In addition, this assay has very low background since it is run in the red visible range that considerably reduces the interference from biological samples. The assay has demonstrated high sensitivity and low interference.

The Amplite™ Colorimetric NADP/NADPH Assay Kit provides a sensi-tive, one-step assay to detect as little as 10 picomoles of NADP(H) in a 100 µL assay volume (100 nM). The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and readily adapted to automation without a separation step. Its signal can be easily read by an absorbance microplate reader at ~575 nm or at the absorbance ratio of ~570 nm to ~605 nm to increase assay sensitivity. Kit 15259 or 15264 are recommended if higher sensitivity is required.

Figure 4.21. NADPH dose response was measured with Amplite™ Colorimetric NADP/NADPH Assay Kit in a 96-well white wall/clear bottom plate using a NOVOStar microplate reader (BMG Labtech). As low as 100 nM (10 pmol/well) of NADPH can be detected with 1 hour incubation time (n=3) while there is no response from NADH.

Amplite™ Fluorimetric NADP/NADPH Assay Kit *Red Fluorescence*


15259 1 kit < - 15 oC

The existing NADP/NADPH assays are run in UV range by absorp-tion. The assays suffer low sensitivity and high interference. This Am-plite™ Fluorimetric NADP/NADPH Assay Kit provides a convenient method for sensitive detection of NADP and NADPH. The enzymes in the system specifically recognize NADP/NADPH in an enzyme cycling reaction that significantly increases detection sensitivity. In addition, this assay has very low background since it is run in the red visible range that significantly reduces the interference resulted from biological samples. There is no need to purify NADP/NADPH from sample mix.

The Amplite™ Fluorimetric NADP/NADPH Assay Kit provides a sensi-tive, one-step assay to detect as little as 1 picomoles of NADP(H) in a 100 µL assay volume (10 nM). The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and read-ily adapted to automation. Its signal can be easily read by either a fluorescence microplate reader at Ex/Em = 540/590 nm or an absorbance microplate reader at ~576 nm. The longer red emission minimizes the interference from the autofluorescence of biological samples.

Figure 4.22. NADPH dose response was measured with Amplite™ Fluorimetric NADP/NADPH Assay Kit in a 96-well black solid plate. As low as 10 nM (1 pmol/well) of NADPH can be detected with 30 min incubation time (n=3) while there is no response from NADH.

168737j1_cat.indd 31 2/4/11 11:53 AM




4

NA

DP/

NA

DPH

Amplite™ Fluorimetric NADP/NADPH Ratio Assay Kit *Red Fluorescence*


15264 1 kit < - 15 oC

The traditional NAD/NADH and NADP/NADPH assays are done by monitoring the changes in NADH or NADPH absorption at 340 nm. These methods suffer low sensitivity and high interference since the assays are done in the UV range that requires expensive quartz microplates. The low sensitivity of the absorption-based NADP/NADPH assays makes the assays difficult to be automated for high throughput screening that often uses small sample size.

This Amplite™ Fluorimetric NADP/NADPH Ratio Assay Kit provides a convenient method for sensitive detection of NADP, NADPH and their ratio. The enzymes in the system specifically recognize NADP/NADPH in an enzyme recycling reaction that significantly increases detection sensitivity. In addition, this assay has very low background since it is run in the red visible range that considerably reduces the sample interference. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format. Its signal can be easily read by either a fluorescence microplate reader at Ex/Em = 530-570/590-600 nm (maximum Ex/Em = 540/590 nm) or an absorbance microplate reader at ~576 nm. This also provides NADP, NADPH extraction buffer, and cell lysis buffer.

Figure 4.23. Total NADPH and NADP, and their extract dose response were measured with Amplite™ Fluorimetric NADP/NADPH Ratio Assay Kit in a 96-well black solid plate. The blank signal was sub-tracted from the values for those wells with the NADPH reactions.

Amplite™ Fluorimetric NADPH Assay Kit *Red Fluorescence*


15262 1 kit < - 15 oC

Nicotinamide adenine dinucleotide (NAD+) and nicotinamide ad-enine dinucleotide phosphate (NADP+) are two important cofactors found in cells. This Amplite™ Fluorimetric NADPH Assay Kit provides a convenient method for the detection of NADPH. The enzymes in the system specifically recognize NADPH in an enzyme recycling reaction. In addition, this assay has very low background since it is run in the red visible range that significantly reduces the interfer-ence from biological samples.

The Amplite™ Fluorimetric NADPH Assay Kit provides a sensitive, one-step assay to detect as little as 100 picomoles of NADPH in a 100 µL assay volume (1 µM). The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and readily adapted to automation. Its signal can be easily read by either a fluorescence microplate reader at Ex/Em = 540/590 nm or an absor-bance microplate reader at ~576 nm. This assay kit has been used for screening enzyme activities that use NADP/NADPH as a cofactor. It has also been used for the sensitive detection of NADPH in cell-based assays. Compared to the other commercial kits, this assay has higher signal/background ratio.

Figure 4.24. NADPH dose response was measured with Amplite™ Fluorimetric NADPH Assay Kit in a 96-well black solid plate. As low as 1µM of NADPH can be detected with 1 hour incubation time (n=3) while there is no response from NADP.

Related Products









168737j1_cat.indd 32 2/4/11 11:53 AM




Related Products






21610 1 kit PhosphoWorks™ Luminometric ATP Assay Kit *Bright Glow*

21609 1 kit PhosphoWorks™ Luminometric ATP Assay Kit *Steady Glow*


4

Phosphate

PhosphoWorks™ Colorimetric MESG Phosphate Assay Kit *UV absorption*


21659 1 kit < - 15 oC

Phosphate is involved in many biological processes. For example, phosphatases, ATPases and several other enzymes catalyze bio-chemical reactions in which inorganic phosphate (Pi) is released from a phosphoester substrate. The detection of many phospho-ester–metabolizing enzymes is difficult because suitable substrates are not available. It is usually necessary to determine inorganic phosphate release using tedious colorimetric assays or radioiso-tope-based methods.

This PhosphoWorks™ Colorimetric MESG Phosphate Assay Kit has been developed for measuring the activity of any Pi-generating enzyme using MESG reagent. The measurement of Pi is based on absorbance change of MESG by phosphate. In the presence of inorganic phosphate, MESG is converted to 2-amino-6-mercapto-7-methlpurine by purine nucleoside phosphorylase with absorption wavelength shift to red. This feature has been used to develop our convenient MESG phosphate assay kit, an alternative to hazard-ous radioactive methods. The MESG substrate gives an absorbance increase at 360 nm on phosphorylysis at pH 6.5-8.5. The assay is shown to quantitate phosphate at the final concentration as low as 0.2 µM. The kit has been used for monitoring ATPase activities. It can also be used for monitoring phosphatase activities.

Figure 4.25. Phosphate dose response was measured with the PhosphoWorks™ Colorimetric MESG Phosphate Assay Kit in a 96-well UV plate. As low as 0.2 µM phosphate can be detected with 30 min incubation time.

PhosphoWorks™ Colorimetric Phosphate Assay Kit *Blue Color*


21665 1 kit < - 15 oC

Cells utilize a wide variety of phosphate and polyphosphate esters as enzyme substrates, second messengers, membrane structural components and vital energy reservoirs. Phosphate is involved in many biological processes. For example, phosphatases, ATPases and several other enzymes catalyze biochemical reactions in which inorganic phosphate (Pi) is released from a phosphoester substrate. The detection of many phosphoester–metabolizing enzymes is difficult because suitable substrates are not available. The tedious radioisotope-based method is usually used to determine inorganic phosphate release.

This PhosphoWorks™ Colorimetric Phosphate Assay Kit has been developed to measure the activity of any Pi-generating enzyme using a modified Malachite Green formulation. It provides sensitive detection of Pi, an alternative to hazardous radioactive methods. The measurement of Pi is based on absorbance change of MG Plus™ in the presence of molybdate. Unlike other Malachite Green formu-lations, this kit gives a completely stable end-point signal that is not prone to precipitation. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format. This assay is non-contin-uous while kit 21660 is a continuous assay. It is also complementary to kit 21659 that uses MESG as a continuous Pi indicator.

Figure 4.26. Phosphate dose response was measured with the PhosphoWorks™ Colorimetric Phos-phate Assay Kit in a 96-well clear plate. As low as 0.1µM phosphate can be detected with 10 minutes incubation time.

168737j1_cat.indd 33 2/4/11 11:53 AM



Cat # Size Storage ConditionCat # Size Storage Condition

34



Pho

spha

te/P

yrop

hosp

hate

4

PhosphoWorks™ Fluorimetric Phosphate Assay Kit *Red Fluorescence*


21660 1 kit < - 15 oC

Cells utilize a wide variety of phosphate and polyphosphate esters as enzyme substrates, second messengers, membrane structural components and vital energy reservoirs. Phosphate is involved in many biological processes. The detection of many phospho-ester–metabolizing enzymes is difficult because suitable substrates are not available. It is usually necessary to determine inorganic phosphate release using tedious colorimetric assays or radioisotope based methods.

This PhosphoWorks™ Fluorimetric Phosphate Assay Kit has been developed to measure the activity of any Pi-generating enzyme us-ing our red fluorescent phosphate sensor. The sensitive detection of Pi is based on the change in the absorbance or fluorescence of the new phosphate sensor. It is an alternative to hazardous radioactive methods and other less sensitive colorimetric assays. Our kit pro-vides all the essential reagents including phosphate sensor, phos-phate standards, and assay buffer. The assay is shown to quantitate phosphate as low as 0.1 μM. It can be used to measure the kinetics of phosphate release from phosphatases by coupling the two enzy-matic reactions. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format.

Figure 4.27. Phosphate dose response was measured with the PhosphoWorks™ Fluorimetric Phosphate Assay Kit in a 96-well black plate. As low as 0.1 µM phosphate can be detected with 1 hour incubation time.

PhosphoWorks™ Fluorimetric Pyrophosphate Assay Kit *Blue Fluorescence*


Pyrophosphate (PPi) are produced by a number of biochemical reactions, such as ATP hydrolysis, DNA and RNA polymerizations, cyclic AMP formation by the enzyme adenylate cyclase and the enzymatic activation of fatty acids to form their coenzyme A esters.

Our PhosphoWroks™ Pyrophosphate Assay Kit provides the most robust spectrophotometric method for the measurement of pyrophosphate. It uses our proprietary fluorogenic pyrophosphate sensor that has its fluorescence intensity proportionally dependent upon the concentration of pyrophosphate. Our assay is much easier and more robust than enzyme-coupling pyrophosphate methods, which require at least two enzymes for their pyrophosphate detec-tions. Due to its direct measurement of pyrophosphate, this kit is ideal for screening inhibition or activities of enzymes that consume or generate pyrophosphate. The assay can be performed in a conve-nient 96-well or 384-well microtiter-plate format. It is an optimized mix-and-read assay. The kit provides all the essential components for assaying pyrophosphate as listed below:

• Assay buffer• PPi sensor• PPi standard

Figure 4.28. Pyrophosphate and phosphate dose response was measured with the PhosphoWorksTM Fluorimetric Pyrophosphate Assay Kit in a 96-well black solid plate. As low as 1 µM (100 picmoles/well) pyrophosphate can be detected with 10 minutes incubation time.

Related Products



21659 1 kit PhosphoWorks™ Colorimetric MESG Phosphate Assay Kit *UV absorption*

21665 1 kit PhosphoWorks™ Colorimetric Phosphate Assay Kit *Blue Color*





168737j1_cat.indd 34 2/4/11 11:53 AM


Enzyme Activity A

ssays

Enzyme Activity Assays 5

168737j1_cat.indd 35 2/4/11 11:53 AM



Enzyme Activity Assays www.aatbio.com

Enz

yme

Activ

ity A

ssay

s

5

enzymatic assay kits at-a-glance*

Enzyme Assays Absorption Fluorescence Luminescence

Acetylcholinesterase 11400 11401 & 11402

Alkaline Phosphatase 11950 11952 ,11953 &11954 11956

Caspase 3/7 13502 & 13503

Catalase 11306

Glucose Oxidase 11300

Glutamate Oxidase 11302

HDAC Activity 13601

Kinase 31001

Lysyl Oxidase 15255

Monoamine Oxidase 11303

MMP-3 13512

MMP 13510 & 13511

Myelopeoxidase 11301

NADPH Regeneration 15265

Peroxidase (HRP) 11551 11552 & 11553 11559

Goat Anti-Mouse IgG-HRP 11540

Goat Anti-Rabbit IgG-HRP 11541

Proteasome 20S Activity 13456

Protease (Generic Activity) 13500 & 13501

Renin 13530

Sphingomyelinase 13620 13621 & 13622

Superoxide Dismutase (SOD) 11305

Xanthine Oxidase 11304




www.aatbio.com Enzyme Activity Assays

Related Products




11402 1 kit Amplite™ Fluorimetric Acetylcholinesterase Assay Kit *Red Fluorescence*



21507 5 mg Thiolite™ Blue


5

Acetylcholinesterase

Amplite™ Colorimetric Acetylcholinesterase Assay Kit


11400 1 kit < - 15 oC

Acetylcholinesterase (AChE) is one of the most crucial enzymes for nerve response and function. AChE degrades the neurotransmitter acetylcholine (ACh) into choline and acetic acid. It is mainly found at neuromuscular junctions and cholinergic synapses in the central nervous system, where its activity serves to terminate the synaptic transmission. AChE inhibitors are among the key drugs approved for Alzheimer’s disease (AD) and myasthenia gravis.

Our Amplite™ Colorimetric Acetylcholinesterase Assay Kit provides a convenient method for the detection of AChE activity. It uses DTNB to quantify the thiocholine produced from the hydrolysis of acetyl-thiocholine by AChE in blood, in cell extracts, and in other solutions. The absorption intensity of DTNB adduct is used to measure the amount of thiocholine formed, which is proportional to the AChE activity. The kit provides a colorimetric one-step assay to detect as little as 0.1 mU AChE in a 100 µL assay volume (1 mU/mL). Its signal can be easily read by an absorbance microplate reader at ~410 nm. The kit is robust and can be used for continuously monitoring AChE activities. It comes with the following components:

• Acetylthiocholine • Acetylcholinesterase standard• DTNB• Assay buffer

Figure 5.1. Acetylcholinesterase dose response was measured in a 96-well clear plate with #11400. As low as 0.1 mU/well of acetylcholinesterase can be detected.

Amplite™ Fluorimetric Acetylcholinesterase Assay Kit *Green Fluorescence*


11401 1 kit < - 15 oC

This kit uses our outstanding Thiolite™ Green to quantify the thio-choline produced from the hydrolysis of acetylthiocholine by AChE in blood, in cell extracts, and in other solutions. Thiolite™ Green is not fluorescent until reacted with a thiol group. It has spectral prop-erties similar to those of fluorescein, making this assay compatible with almost every fluorescence instrument. The fluorescence inten-sity of Thiolite™ Green is used to measure AChE activity. Compared to the existing thiol probes (e.g., mBBr and bBBr), Thiolite™ Green is much more sensitive.

The Amplite™ Fluorimetric Acetylcholinesterase Assay Kit provides an ultrasensitive fluorometric one-step assay to detect as little as 0.01mU AChE in a 100 µL assay volume (0.1 mU/mL). The assay can be performed in a convenient 96-well or 384-well microtiter-plate format. Its signal can be easily read by a fluorescence microplate reader at Ex/Em = 490/520 nm. Our Amplite™ Fluorimetric Acetyl-cholinesterase Assay Kit provides the most sensitive method for the detection of AChE activity. It is robust to use and comes with the following components:

• Thiolite™ Green sensor• Acetylthiocholine • Acetycholinesterase standard• Assay buffer

Figure 5.2. Acetylcholinesterase dose response was measured in a 96-well black solid plate with 11401. As low as 0.01 mU/well of acetylcholinesterase can be detected with 20 minutes incubation time (n=3).

168737j1_cat.indd 37 2/4/11 11:53 AM




5

Ace

tylc

holin

este

rase

/Alk

alin

e Ph

osph

atas

e

Amplite™ Fluorimetric Acetylcholinesterase Assay Kit *Red Fluorescence*


11402 1 kit < - 15 oC

Acetylcholinesterase (AChE) is one of the most crucial enzymes for nerve response and function. AChE degrades the neurotransmitter acetylcholine (ACh) into choline and acetic acid. It is mainly found at neuromuscular junctions and cholinergic synapses in the central nervous system, where its activity serves to terminate the synaptic transmission. AChE inhibitors are among the key drugs approved for Alzheimer’s disease (AD) and myasthenia gravis.

Our Amplite™ Fluorimetric Acetylcholinesterase Assay Kit provides one of the most sensitive methods for detecting AChE activity or screening AChE inhibitors in red florescence window. The kit uses Amplite™ Red to quantify the choline produced from the hydro-lysis of acetylcholine by AChE through choline oxidase-mediated enzyme coupling reactions. It can be used for monitoring and quantifying the AChE activity in blood, cell extracts or other solu-tions. The fluorescence intensity of Amplite™ Red is used to measure the amount of choline formed, which is proportional to the AChE activity. The kit is an optimized “mix and read” assay that provides a simple one-step fluorimetric assay to detect as little as 0.01 mU AchE in a 100 µL assay volume (0.1 mU/mL). Its signal can be easily read with a fluorescence microplate reader at Ex/Em = ~540/590 nm or an absorbance microplate reader at ~575 nm.

Figure 5.3. Acetylcholinesterase dose response was measured in a 96-well black solid plate with Amplite™ Fluorimetric Acetylcholinesterase Assay Kit (11402). As low as 0.01 mU/well (0.1mU/mL) of acetylcholinesterase can be detected with 20 minutes incubation time (n=3).

Related Products


11400 1 kit Amplite™ Colorimetric Acetylcholinesterase Assay Kit



11952 1 kit Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit *Blue Fluorescence*

11953 1 kit Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit *Green Fluorescence*

11954 1 kit Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit *Red Fluorescence*


Amplite™ Colorimetric Alkaline Phosphatase Assay Kit *Yellow Color*


11950 1 kit < - 15 oC

Alkaline phosphatase (ALP) (EC 3.1.3.1) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The pro-cess of removing the phosphate group is called dephosphorylation. An important use of alkaline phosphatase is as a label for enzyme immunoassays. Alkaline phosphatase is a highly sensitive enzyme for ELISA, immuno-histochemical as well as Northern, Southern and Western blot applications. It is widely used in various biological as-says (in particular, immunoassays) and ELISA-based diagnostics.

Our Amplite™ Colorimetric Alkaline Phosphatase Assay Kit uses pNPP, a chromogenic phosphatase substrate, to quantify alkaline phosphatase activity in solutions, in cell extracts, and on solid surfaces (such as PVDF membranes). The kit provides an optimized “mix and read” assay protocol which is compatible with HTS liquid handling instruments. Its signal can be easily read by an absorbance microplate reader at around 400 nm. This Amplite™ Colorimetric Alkaline Phosphatase Assay Kit can be performed in a convenient 96-well or 384-well and comes with the following components:

• pNPP substrate• Alkaline phosphatase• Assay buffer

Figure 5.4. Alkaline phosphatase dose response was measured with the Amplite™ Colorimetric Alkaline Phosphatase Assay Kit in a white/clear bottom 96-well plate using a NOVOstar microplate reader (BMG Labtech). As low as 0.3 mU/mL of alkaline phosphatase can be detected with 30 minutes incubation time (n=3).

168737j1_cat.indd 38 2/4/11 11:53 AM




5

Alkaline Phosphatase

Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit *Blue Fluorescence*


11952 1 kit < - 15 oC

This Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit uses our MUP Plus™-based coumarin substrate. Similar to MUP, MUP Plus™ is sensitive to phosphatase-induced hydrolysis, giving the haloge-nated coumarin that possesses intense blue fluorescence. Its almost identical spectral properties to those of MUP enables MUP Plus™ substrates readily compatible with many fluorescence instrument systems equipped with MUP settings. Compared to MUP, MUP Plus™ gives the coumarin fluorophore that has substantially lower pKa, making the MUP Plus™ assay much less pH-dependent.

Our Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit uses our MUP Plus™, a fluorogenic phosphatase substrate, to quantify alkaline phosphatase activity in solutions, in cell extracts, and on solid surfaces (such as PVDF membranes). It can be performed in a convenient 96-well or 384-well microtiter-plate format. Its signal can be easily read by a fluorescence microplate reader at Ex/Em = ~360/450 nm. The kit provides an optimized “mix and read” assay protocol and comes with the following components:

• MUP Plus™ substrate• Alkaline phosphatase• Assay buffer

Figure 5.5. Alkaline phosphatase dose response was measured with the Amplite™ Fluorimetric Alka-line Phosphatase Assay Kit in a solid black 96-well plate using a Gemini microplate reader (Molecular Devices). As low as 0.1 mU/mL of alkaline phosphatase can be detected with 30 minutes incubation time (n=3).

Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit *Green Fluorescence*


11953 1 kit < - 15 oC

Alkaline phosphatase (ALP) (EC 3.1.3.1) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. An impor-tant use of alkaline phosphatase is as a label for enzyme immunoas-says. Alkaline phosphatase is a highly sensitive enzyme for ELISA, immuno-histochemical as well as Northern, Southern and Western blot applications. It is widely used in various biological assays (in particular, immunoassays) and ELISA-based diagnostics.

Our Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit uses our FDP, a fluorogenic phosphatase substrate, to quantify the alkaline phosphatase activity in solutions, in cell extracts, and on solid surfaces (such as PVDF membranes). It can be performed in a convenient 96-well or 384-well microtiter-plate format. Its signal can be easily read by a fluorescence microplate reader at Ex/Em = ~490/525 nm. The kit provides an optimized “mix and read” assay protocol with the following components:

• FDP substrate• Alkaline phosphatase• Assay buffer

Figure 5.6. Alkaline phosphatase dose response was measured with the Amplite™ Fluorimetric Alka-line Phosphatase Assay Kit in a solid black 96-well plate using a Gemini microplate reader (Molecular Devices). As low as 0.1 mU/mL of alkaline phosphatase can be detected with 30 minutes incubation time (n=3).

Related Products



11628 10 mg CF-MUP, sodium salt *Superior alternative to MUP*


11600 5 mg FDP [Fluorescein diphosphate, tetraammonium salt]

21665 1 kit PhosphoWorks™ Colorimetric Phosphate Assay Kit *Blue Color*



168737j1_cat.indd 39 2/4/11 11:53 AM




5

Alk

alin

e Ph

osph

atas

e

Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit *Red Fluorescence*


11954 1 kit < - 15 oC

Alkaline phosphatase is widely used in various biological assays (in particular, immunoassays) and ELISA-based diagnostics. Our Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit uses our SunRed™-based substrate. The weakly fluorescent SunRed™ phos-phate is sensitive to phosphatase-induced hydrolysis, giving the SunRed™ fluorophore that possesses intense red fluorescence. Upon phosphatase-induced hydrolysis, the SunRed™ phosphate solution has its absorption blue-shifted more than 100 nm. The maximum absorption of SunRed™ fluorophore at 633 nm makes this substrate an ideal NIR probe that can be readily detected with many fluores-cence instrument systems often equipped with Cy5 settings.

Based on the near infrared fluorescence of SunRed™ fluorophore, the signal can be easily read by a fluorescence microplate reader at Ex/Em = ~630/660 nm. The kit has been used for the high through-put screening of protein phosphatase inhibitors due to its low inter-ference from biological sample. It can be performed in a convenient 96-well or 384-well microtiter-plate format and comes with the following components:

• SunRed™ substrate• Alkaline phosphatase• Assay buffer

Figure 5.7. Alkaline phosphatase dose response was measured with the Amplite™ Fluorimetric Alka-line Phosphatase Assay Kit in a solid black 96-well plate using a Gemini microplate reader (Molecular Devices). As low as 3 mU/mL of alkaline phosphatase can be detected with 60 minutes incubation time (n=3).

Amplite™ Luminometric Alkaline Phosphatase Assay Kit


11956 1 kit < - 15 oC

Alkaline phosphatase (ALP) (EC 3.1.3.1) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The pro-cess of removing the phosphate group is called dephosphorylation. An important use of alkaline phosphatase is as a label for enzyme immunoassays.

Our Amplite™ Luminometric Alkaline Phosphatase Assay Kit uses D-luciferin phosphate as the luminogenic phosphatase substrate to quantify alkaline phosphatase activity in solutions and in cells. D-luciferin phosphate is not recognized by luciferase until its phosphate group is removed to give luciferin. The kit provides an optimized “mix and read” assay protocol which is compatible with HTS liquid-handling instruments. This Amplite™ Luminometric Al-kaline Phosphatase Assay Kit can be readily performed in a 96-well or 384-well microtiter-plate format. Its signal can be easily read by luminescence microplate readers. The high sensitivity makes the kit ideal for the assays that require low detection limit. The kit is conve-nient to use and comes with the following components:

• D-luciferin phosphate• Alkaline phosphatase• Assay buffer

Figure 5.8. Alkaline phosphatase dose response was measured with the Amplite™ Luminometric Alkaline Phosphatase Assay Kit in a 96-well white plate using a NOVOstar microplate reader (BMG Labtech). As low as 0.01 mU/mL of alkaline phosphatase can be detected with 20 minutes incubation time (n=3).

Related Products


11950 1 kit Amplite™ Colorimetric Alkaline Phosphatase Assay Kit *Yellow Color*



11952 1 kit Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit *Blue Fluorescence*

11953 1 kit Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit *Green Fluorescence*



168737j1_cat.indd 40 2/4/11 11:53 AM




5

Caspase 3/7

Amplite™ Fluorimetric Caspase 3/7 Assay Kit *Blue Fluorescence*


13502 1 kit < - 15 oC

Caspases play important roles in apoptosis and cell signaling. The activation of Caspase 3/7 (CPP32/apopain) is important for the initi-ation of apoptosis. Caspase 3/7 is also identified as a drug-screening target. Caspase inhibitors have anti-cancer and other pharmalogical potentials. It has been proven that Caspase 3/7 has substrate selec-tivity for the peptide sequence Asp-Glu-Val-Asp (DEVD).

Our Amplite™ Fluorimetric Caspase 3/7 Assay Kit uses Ac-DEVD-AMC as a fluorogenic indicator for Caspase 3/7 activity. AMC peptides are almost non-fluorescent. Cleavage of AMC peptides by Caspase 3/7 generates strongly fluorescent AMC that is fluorimetri-cally monitored at 440-460 nm with excitation of 340-350 nm. It can be used to continuously measure the activities of Caspase 3/7 in cell extracts and purified enzyme preparations with a fluorescence microplate reader or fluorometer. The kit comes with the following components:

• AMC caspase substrate• Ac-DEVD-CHO caspase inhibitor• DTT• Assay buffer

Figure 5.9. Detection of Caspase 3/7 Activity in Jurkat cells. Jurkat cells were seeded on the same day at 80,000 cells/well/90 µL in a 96-well black wall/clear bottom Costar plate. The cells were treated with or without 20 µM of camptothecin for 5 hours, and/or 5 µM of the caspase inhibitor AC-DEVD-CHO for 10 minutes. The caspase 3/7 assay solution (100 µL/well) was added and incubated at room tempera-ture for 1 hour. The fluorescence intensity was measured at Ex/Em = 350/450 nm.

Amplite™ Fluorimetric Caspase 3/7 Assay Kit *Green Fluorescence*


13503 1 kit < - 15 oC

Our Amplite™ Fluorimetric Caspase 3/7 Assay Kit uses (Z-DEVD)2 R110 as fluorogenic indicator for assaying caspase 3/7 activity. R110-derived caspase substrates are probably the most sensitive indicators used for the fluorimetric detection of various caspase activities. R110 peptides are colorless and non-fluorescent. Cleav-age of R110 peptides by caspases generates strongly fluorescent R110 that can be monitored fluorimetrically at 510-530 nm with excitation at 488 nm, the most common excitation light source. The increase in fluorescence of caspase-induced R110 hydrolysis is proportional to the activities of caspases.

This kit can be used to continuously measure the activities of caspase 3/7 in cell extracts and purified enzyme preparations with a fluorescence microplate reader or fluorometer. Many labs have used this kit for screening aspase 3/7 inhibitors. The kit comes with the following components:

• (Z-DEVD)2R110 substrate• Ac-DEVD-CHO inhibitor• DTT• Assay buffer

Figure 5.10. Detection of caspase 3/7 Activity in Jurkat cells. Jurkat cells were seeded on the same day at 80,000 cells /well/90 µL in a 96-well black wall/clear bottom costar plate. The cells were treated with or without 20 µM of camptothecin for 5 hours, and/or 5 µM of the caspase inhibitor AC-DEVD-CHO for 10 minutes. The caspase 3/7 assay solution (100 µL/well) was added and incubated at room tempera-ture for 1 hour. The fluorescence intensity was measured at Ex/Em = 490/525 nm.

Related Products


13401 5 mg Ac-DEVD-AFC

13402 5 mg Ac-DEVD-AMC

13406 1 mg FITC-C6-DEVD-FMK

13455 1 mg (Ac-ANW)2 -R110

13431 1 mg (Ac-IETD)2-R110

13430 1 mg (Z-DEVD)2-R110

13452 1 mg Suc-LLVY-D-Aminoluciferin

168737j1_cat.indd 41 2/4/11 11:53 AM



Enzyme Activity Assays www.aatbio.com C

atal

ase/

Glu

cose

Oxi

dase

Related Products


11000 1 kit Amplite™ Colorimetric Acetylcholinesterase Assay Kit




40005 1 kit Amplite™ Glucose Quantitation Kit

11503 1 kit Amplite™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Green Fluorescence*


5

Amplite™ Fluorimetric Catalase Assay Kit *Red Fluorescence*


11306 1 kit < - 15 oC

Catalase is a common antioxidant heme-containing redox enzyme found in nearly all living organisms that are exposed to oxygen. The enzyme is concentrated in the peroxisome subcellular organelles. Hydrogen peroxide is an ROS that is a toxic product of normal aero-bic metabolism and pathogenic ROS production involving oxidase and superoxide dismutase reactions. By preventing the excessive buildup of H2O2, catalase allows important cellular processes which produce H2O2 as a by-product to take place safely.

The Amplite™ Fluorimetric Catalase Assay Kit provides a quick and sensitive method for the measurement of catalase activity. It can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. Catalase reacts with H2O2 to produce water and oxygen (O2). Amplite™ Red also reacts with H2O2 to generate a red fluorescent product. Therefore the reduction in fluorescence intensity is propor-tional to catalase activity. The Amplite™ Red substrate used in the assay enables a dual recordable mode. The fluorescent signal can be easily read by either a fluorescence microplate reader at Ex/Em = 540/590 nm or an absorbance microplate reader at ~576 nm. With the Amplite™ Fluorimetric Catalase Assay Kit, we have detected as little as 30 mU/mL catalase in a 100 µL reaction volume.

Amplite™ Fluorimetric Glucose Oxidase Assay Kit*Red Fluorescence*


11300 1 kit < - 15 oC

The glucose oxidase is a dimeric protein that catalyzes the oxidation of beta-D-glucose into hydrogen peroxide and D-glucono-1,5-lac-tone, which is hydrolyzed to gluconic acid. It is widely used for the determination of glucose in body fluids and in removing residual glucose and oxygen from beverages, food and other agricultural products. Furthermore, glucose oxidase is commonly used in bio-sensors to detect glucose.

The Amplite™ Glucose Oxidase Assay Kit provides a quick and sensi-tive method for the measurement of glucose oxidase in solution. It can be performed in a convenient 96-well or 384-well microtiter-plate format and is easily adapted to automation without a separa-tion step. The kit uses our Amplite™ Red substrate which enables a dual recordable mode. The fluorescent signal can be easily read by either a fluorescence microplate reader at Ex/Em = 540/590 nm or an absorbance microplate reader at ~576 nm. With the Amplite™ Fluorimetric Glucose Oxidase Assay Kit, we have detected as little as 0.05 mU/mL glucose oxidase in a 100 µL reaction volume. The kit comes with the following components:

• Amplite Red™ HRP substrate• HRP enzyme• Assay buffer

Figure 5.11. Catalase dose response was measured with Amplite™ Fluorimetric Catalase Assay Kit in a 96 well black solid plate using a Gemini fluorescence microplate reader (Molecular Devices). As low as 30 mU/mL catalase was detected with 30 minutes incubation time (n=3).

Figure 5.12. Glucose oxidase dose response was measured with Amplite™ Glucose Oxidase Assay Kit in a 96-well black solid plate using a Gemini fluorescence microplate reader (Molecular Devices). As low as 0.05 mU/mL glucose oxidase was detected with 30 minutes incubation time (n=3).

168737j1_cat.indd 42 2/4/11 11:53 AM




5

Glutam

ate Oxidase/H

DAC

Amplite™ Fluorimetric Glutamate Oxidase Assay Kit *Red Fluorescence*


11302 1 kit < - 15 oC

Glutamate oxidase belongs to the family of oxidoreductases, specifi-cally those acting on the CH-NH2 group of donors with oxygen as an acceptor. It is an enzyme that specifically catalyzes the oxidative de-amination of L-glutamate in the presence of water and oxygen with the formation of o-ketoglutarate, ammonia, and hydrogen peroxide.

The Amplite™ Fluorimetric Glutamate Oxidase Assay Kit provides a quick and ultrasensitive method for the measurement of glutamate oxidase in solution and in cell lysates. In the assay, L-glutamic acid is oxidized to α-ketoglutarate, NH3 and H2O2 by glutamate oxidase. L-Alanine and L-glutamate-pyruvate transaminase are included in the reaction, resulting in multiple cycles of the initial reaction, thus significantly amplifying the production of H2O2. The kit uses our Amplite™ Red substrate which enables a dual recordable mode. The fluorescent signal can be easily read by either a fluorescence micro-plate reader at Ex/Em = 540/590 nm or an absorbance microplate reader at ~576 nm. With the Amplite™ Glutamate Oxidase Assay kit, we have detected as little as 40 μU/mL glutamate oxidase in a 100 µL reaction volume. It can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step.

Amplite™ Fluorimetric HDAC Activity Assay Kit *Green Fluorescence*


13601 1 kit < - 15 oC

Histone deacetylases (HDAC) are a class of enzymes that remove acetyl groups from a ε-N-acetyl lysine amino acid on a histone. Deacetylation restores the positive electric charge of the lysine amino acids, which increases the histone’s affinity to the negatively charged phosphate backbone of DNA. This process generally down-regulates DNA transcription by blocking the access of transcription factors. HDAC inhibitors are being studied as a treatment for cancer.

Our Amplite™ Fluorimetric HDAC Activity Assay Kit provides a quick, convenient, and sensitive method for the detection of HDAC activity. This kit uses our non-peptide HDAC Green™ substrate that is much more sensitive than the peptide-based HDAC substrates such as Ac-RGK(Ac)-R110, Ac-RGK(Ac)-AMC and Ac-RGK(Ac)-AFC. In addition, HDAC Green™ substrate is also much more resistant to protease hydrolysis than other commercial peptide-based HDAC substrates. Our kit can be used for measuring HDAC activity in cell lysates or HDAC inhibitor screening with cell extracts or purified enzymes. The long wavelength emission of the HDAC Green™ substrate makes the assay less interfered from compounds and cell components. HDAC activity is monitored with excitation at 490 nm and emission at 525 nm.

Related Products



11300 1 kit Amplite™ Fluorimetric Glucose Oxidase Assay Kit *Red Fluorescence*






Figure 5.13. Glutamate oxidase dose response was measured with Amplite™ Fluorescence Glutamate Oxidase Assay Kit on a 96-well black solid plate using a Gemini fluorescence microplate reader (Molecular Devices). As low as 40 µU/mL glutamate oxidase was detected with 30 minutes incubation time (n=3).

Figure 5.14. HDAC activity in HeLa nuclear extract measured with Amplite™ Fluorimetric HDAC Activ-ity Assay Kit (in blue) was compared with Vendor X (in red) and Vendor Y (in green), both of which use Ac-RGK(Ac)-R110 peptide substrate. The signal/background ratio of the HDAC activity measured with Amplite™ Fluorimetric HDAC Activity Assay Kit is more than10 times higher than those of Vendors X and Y.

168737j1_cat.indd 43 2/4/11 11:53 AM



5

Kin

ase/

Lysy

l Oxi

dase


Amplite™ Universal Fluorimetric Kinase Assay Kit *Red Fluorescence*


31001 1 kit < - 15 oC

Protein kinases are the enzymes that transfer a phosphate group from a phosphate donor to an acceptor amino acid in a substrate protein. Kinases are of great interest to researchers involved in drug discovery. Most of the commercial protein kinase assay kits are based on monitoring either the phosphopeptide formation or the ATP depletion. For the kinase assay kits that are based on the detection of phosphopeptides, one has to spend time and efforts to identify an optimized peptide substrate while the ATP depletion method suffers various interferences due to the use of luciferase that are inhibited or activated by various biological compounds.

The Amplite™ Universal Fluorimetric Kinase Assay Kit is based on monitoring ADP formation, which is directly proportional to en-zyme phosphotransferase activity and is measured fluorimetrically. This enzyme-coupled kit provides a fast, simple, and homogeneous assay to measure kinase activities. It is a non-radioactive and no wash method to detect the amount of ADP produced from enzyme reaction. Its characteristics of high sensitivity (<0.3 μM ADP) and broad ATP tolerance (1-300 μM) make it an ideal kit for determining kinase Michaelis-Menten kinetics and for screening and identifying kinase inhibitors. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format.

Figure 5.15. ADP dose response was measured with the Amplite™ Universal Fluorimetric Kinase Assay Kit in a 384-well black solid plate. As low as 0.3 µM ADP can be detected with 30 minutes incubation time.

Amplite™ Fluorimetric Lysyl Oxidase Assay Kit *Red Fluorescence*


15255 1 kit < - 15 oC

Lysyl oxidase is an extracellular enzyme that catalyzes formation of aldehydes from lysine residues in collagen and elastin precur-sors. These aldehydes are highly reactive, and undergo spontane-ous chemical reactions with other lysyl oxidase-derived aldehyde residues or with unmodified lysine residues. The chemical reactions result in cross-linking collagen and elastin, which is essential for stabilization of collagen fibrils and for the integrity and elasticity of mature elastin. The activity of Lysyl oxidase in biological samples is traditionally assessed by tritium release end-point assays using radio isotope labeled collagen or elastin substrates.

The Amplite™ Fluorimetric Lysyl Oxidase Assay Kit offers a sensi-tive fluorescent assay for detecting the activity of lysyl oxidase. It utilizes a proprietary LOX substrate that releases hydrogen peroxide detected using our Amplite™ ADHP substrate in HRP-coupled reac-tions. This method allows the detection of sub ng/mL lysyl oxidase and is much more sensitive than the currently available assays. It eliminates the interference that occurs in some biological samples and can be readily used to detect lysyl oxidase activity in cell ex-tracts or solutions. Its signal can be easily read by either a fluores-cence microplate reader at Ex/Em = 540/590 nm or an absorbance microplate reader at ~576 nm.

Figure 5.16. Lysyl oxidase dose response was measured on a 96-well black solid plate with the Amplite™ Fluorimetric Lysyl Oxidase Assay Kit. As low as 40 ng of lysyl oxidase can be detected with 30 minutes incubation time (n=3).

Related Products












5

Monoam

ine Oxidase/M

MP-3

Amplite™ Fluorimetric Monoamine Oxidase Assay Kit *Red Fluorescence*


11303 1 kit < - 15 oC

Monoamine oxidases (MAO) are a family of flavin-containing amine oxidoreductases that catalyze the oxidation of monoamines. They are found bound to the outer membrane of mitochondria in numer-ous tissues including liver, intestinal mucosa, and nerves. In humans there are two types of MAO: MAO-A and MAO-B. MAO-A is particu-larly important in the metabolism of monoamines ingested in food. MAOs play a major role in the inactivation of neurotransmitters. MAO dysfunction has been associated with depression, schizo-phrenia, substance abuse, attention deficit disorder, migraines, and irregular sexual maturation.

The Amplite™ Monoamine Oxidase Assay Kit provides a quick and sensitive method for the measurement of monoamine oxidase and semicarbazide-sensitive amine oxidase (SSAO) activities in blood samples and other biological samples. The kit uses our Amplite™ Red substrate which enables a dual recordable mode. The signal can be easily read by either a fluorescence microplate reader at Ex/Em = 540/590 nm or an absorbance microplate reader at ~576 nm. With the Amplite™ Monoamine Oxidase Assay Kit, we have detected as little as 10 μU/mL SSAO in a 100 µL reaction volume. It can be per-formed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation.

Amplite™ Fluorimetric MMP-3 Activity Assay Kit *Green Fluorescence*


13512 1 kit < - 15 oC

MMP-3 (Matrix Metalloproteinase-3), also known as stromelysin-1, is involved in wound repair, atherosclerosis and tumors. Our Amplite™ Fluorimetric MMP-3 Activity Assay Kit uses a Tide Fluor™ 2 (TF2)/Tide Quencher™ 2 (TQ2) fluorescence resonance energy transfer (FRET) peptide as the MMP-3 activity indicator. In the intact FRET peptide, the fluorescence of TF2 is quenched by TQ2. Upon cleavage into two separate fragments by MMP-3, the green fluorescence of TF2 is recovered.

This kit is designed to monitor the activity of an MMP-3 enzyme. The poptide sequence used in the kit is more selective for MMP-3 hydro-lysis than other MMP enzymes. It can also be used to screen MMP-3 inhibitors when a purified MMP-3 enzyme is used. With excellent fluorescence quantum yield and longer wavelength, TF2 shows less interference from autofluorescence of test compounds and cellular components and is much more sensitive than an EDANS/Dabcyl FRET substrate. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format. Its signal can be easily read by a fluorescence microplate reader at Ex/Em = 490/525 nm. The perfect excitation of TF2 at 488 nm makes the assay readily readable with almost all the common fluorescence instruments equipped with Argon laser and FITC filter set.

Figure 5.18. Dose response of MMP-3 enzyme activity was measured with a NOVOstar microplate reader (BMG Labtech). As low as 1 ng/well MMP-3 was detected with 60 min incubation time (n=3). Note: MMP-3 from different sources will vary in its endogenous activity.

Related Products


13500 1 kit Amplite™ Universal Fluorimetric Protease Activity Assay Kit *Green Fluorescence*

13501 1 kit Amplite™ Universal Fluorimetric Protease Activity Assay Kit *Red Fluorescence*

13440 5 mg Casein, FITC-conjugated

13441 5 mg Casein, TAMRA-conjugated

13520 1 mg MMP Green™ substrate

13521 1 mg MMP Red™ substrate

13528 1 mg MMP-3 Green™ substrate

Figure 5.17. SSAO dose response was measured with Amplite™ Monoamine Oxidase Assay Kit in a 96-well black solid plate using a Gemini fluorescence microplate reader (Molecular Devices). As low as 10 µU/mL SSAO was detected with 30 minutes incubation time (n=3).

168737j1_cat.indd 45 2/4/11 11:54 AM




5

MM

P Ac

tivity

Amplite™ Universal Fluorimetric MMP Activity Assay Kit *Green Fluorescence*


13510 1 kit < - 15 oC

The matrix metalloproteinases (MMPs) constitute a family of zinc-dependent endopeptidases that function within the extracellular matrix. These enzymes are responsible for the breakdown of con-nective tissues and are important in bone remodeling, the menstru-al cycle, and repair of tissue damage. While the exact contribution of MMPs to certain pathological processes is difficult to assess, MMPs appear to play a key role in the development of arthritis as well as in the invasion and metastasis of cancer.

Our Amplite™ Universal Fluorimetric MMP Activity Assay Kit uses a Tide Fluor™ 2 (TF2)/Tide Quencher™ 2 (TQ2) fluorescence resonance energy transfer (FRET) peptide as a generic MMP activity indicator. It is designed to check the general activity of an MMP enzyme and to screen MMP inhibitors. In the intact FRET peptide, the fluorescence of TF2 is quenched by TQ2. After cleaved into two separate frag-ments by MMPs, the fluorescence of TF2 is recovered. With excellent fluorescence quantum yield and longer wavelength, TF2 probe is much more sensitive than an EDANS/Dabcyl FRET substrate. Its signal can be easily read by a fluorescence microplate reader at Ex/Em = 490/525 nm.

Amplite™ Universal Fluorimetric MMP Activity Assay Kit *Red Fluorescence*


13511 1 kit < - 15 oC

This Amplite™ Universal Fluorimetric MMP Activity Assay Kit uses a Tide Fluor™ 3 (TF3)/Tide Quencher™ 3 (TQ3) fluorescence resonance energy transfer (FRET) peptide as a MMP substrate. In the intact FRET peptide, the fluorescence of TF3 is quenched by TQ3. Upon cleavage into two separate fragments by MMPs, the fluorescence of TF3 is recovered. This kit is designed to check the general activity of a MMP enzyme. It can also be used to screen MMP inhibitors when a purified MMP enzyme is used.

With excellent fluorescence quantum yield and longer wavelength, TF3 shows less interference from autofluorescence of test com-pounds and cellular components and is much more sensitive than an EDANS/Dabcyl FRET substrate. Its signal can be easily read by a fluorescence microplate reader at Ex/Em = 540/590 nm. The pH-independent fluorescence of TF3 makes the assay reading available for the whole physiological pH range. The high photostability of TF3 might make the TF3/TQ3 FRET peptide a useful imaging probe. Many labs have used this kit for the high throughput screening of MMP inhibitors as potential anticancer drug candidates. This assay might be also used for monitoring cancer cells.

Figure 5.19. Detection of MMPs activity using Amplite™ Universal Fluorimetric MMP Activity Assay Kit. The fluorescence signal was monitored one hour after the start of the reaction by using a NOVOStar microplate reader with a filter set of Ex/Em = 490/525 nm. The reading from all wells was subtracted with the reading from substrate control, which contains MMP Green™ substrate but no MMPs. The MMP Green™ substrate can detect the activity of sub-nanogram of all MMPs (n=3).

Figure 5.20. Detection of MMPs activity using Amplite™ Universal Fluorimetric MMP Activity Assay Kit. The fluorescence signal was monitored one hour after the start of the reaction. The reading from all wells was subtracted with the reading from substrate control, which contains MMP Red™ substrate but no MMPs. The MMP Red™ substrate can detect the activity of sub-nanogram of all MMPs (n=3).

Related Products


13512 1 kit Amplite™ MMP-3 Activity Assay Kit *Green Fluorescence*

13510 1 kit Amplite™ Universal Fluorimetric MMP Activity Assay Kit *Green Fluorescence*

13501 1 kit Amplite™ Universal Fluorimetric Protease Activity Assay Kit *Red Fluorescence*

13440 5 mg Casein, FITC-conjugated

13520 1 mg MMP Green™ substrate

13521 1 mg MMP Red™ substrate

13528 1 mg MMP-3 Green™ substrate

168737j1_cat.indd 46 2/4/11 11:54 AM




5

Myelopeoxidase/N

AD

PH

Amplite™ Fluorimetric Myelopeoxidase Assay Kit *Red Fluorescence*


11301 1 kit < - 15 oC

Myeloperoxidase (MPO), most abundantly present in neutrophils and monocytes, is a green hemoprotein having peroxidase activity. It catalyzes the reaction of hydrogen peroxide and halide ions to form cytotoxic acids and other intermediates; and plays an impor-tant role in the oxygen-dependent killing of tumor cells and micro-organisms. MPO deficiency is a hereditary deficiency of the enzyme, which predisposes to immune deficiency. There are considerable interests in the development of therapeutic MPO inhibitors.

Our Amplite™ Myeloperoxidase Assay Kit provides a quick and sen-sitive method for the measurement of myeloperoxidase in solution and in cell lysates. It can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. The kit uses our Amplite™ Red substrate which enables a dual recordable mode. The signal can be easily read by either a fluorescence microplate reader at Ex/Em = 540/590 nm or an absorbance microplate reader at ~576 nm. With the Amplite™ Myeloperoxidase Assay Kit, we have detected as little as 5 ng/mL myeloperoxidase in a 100 µL reaction volume. The kit can be auto-mated for high throughput screenings of MPO inhibitors.

ReadiUse™ NADPH Regenerating Kit


15265 1 kit < - 15 oC

NADPH provides the reducing equivalents for biosynthetic reac-tions and for oxidation-reduction involved in protection against the toxicity of ROS (reactive oxygen species). NADPH is also used for anabolic pathways, such as lipid synthesis, cholesterol synthesis and fatty acid chain elongation. It is a necessary cofactor in many xenobiotic metabolism reactions. In chloroplasts, NADP is reduced to NADPH by ferredoxin-NADP reductase in the last step of the electron chain in photosynthesis reactions. The NADPH produced is used as reducing power for the biosynthetic reactions in the Calvin cycle of photosynthesis. Many oxidoreductases and all ligases use NADPH as coenzyme. NADPH is required for the measurement of oxidase activity catalyzed by P450s, FMOs, NADPH-P450 reductase, and many other oxidase enzymes.

AAT Bioquest’s RediUse™ NADPH Regenerating Kit provides two ready-to-use solutions to regenerate NADPH by a simple mixing. This kit can be used for all NADPH-requiring oxidase assays (cDNA-expressed enzymes and liver fractions). About 300-500 enzyme assays can be performed using this kit. The total number of assays that can be performed depends on one’s experimental design.

Related Products









Figure 5.21. Myeloperoxidase dose response was measured with Amplite™ Myeloperoxidase Assay Kit in a 96-well black solid plate using a Gemini fluorescence microplate reader (Molecular Devices).As low as 5 ng/mL myeloperoxidase was detected with 30 minutes incubation time (n=3).

Figure 5.22. The chemical structures of NADP and NADPH.




5

Per

oxid

ase

Amplite™ Colorimetric Peroxidase Assay Kit *Blue Color*


11551 1 kit < - 15 oC

Horseradish Peroxidase (HRP) is a small molecule (MW ~40 KD) that is widely used in a variety of biological detections. HRP conjugates are extensively used as secondary detection reagents in ELISAs, im-muno-histochemical techniques as well as Northern, Southern and Western blot analyses. Due to its small size, HRP rarely causes steric hindrance problem with the antibody/antigen complex formation. It is usually conjugated to an antibody in a 4:1 ratio. Additionally, HRP is inexpensive compared to other labeling enzymes.

This kit uses Amplite™ Blue, our chromogenic HRP substrate that is much more sensitive to both H2O2 and peroxidase than other chromogenic peroxidase substrates such as TMB, ABTS, OPD and K-Blue. Amplite™ Blue generates a highly absorptive material that has maximum absorption of 664 nm. This near infrared absorption minimizes the background absorption often caused by the auto-absorption of biological samples. Its signal can be easily read by an absorbance microplate reader at ~664 nm. The kit comes with the following components:

• Amplite™ Blue substrate• HRP and H2O2

• Assay buffer

Amplite™ Fluorimetric Peroxidase Assay Kit *Near Infrared Fluorescence*


11553 1 kit < - 15 oC

Horseradish Peroxidase (HRP) is a small molecule (MW ~40 KD) that is widely used in a variety of biological detections. HRP conjugates are extensively used as secondary detection reagents in ELISAs, im-muno-histochemical techniques as well as Northern, Southern and Western blot analyses. Due to its small size, HRP rarely causes steric hindrance problem with the antibody/antigen complex formation.

We offer this quick HRP assay in a one-step, homogeneous, no wash assay system. This kit uses our fluorogenic Amplite™ IR HRP substrate to quantify peroxidase in solutions. The kit can be used for ELISAs, characterizing kinetics of enzyme reaction and high throughput screenings. It provides an optimized “mix and read” assay protocol. This Amplite™ Fluorimetric Peroxidase Assay Kit can detect as low as 1 mU/mL of HRP. It can be performed in a conve-nient 96-well or 384-well microtiter-plate format. Its signal can be easily read by either a fluorescence microplate reader at Ex/Em = 640/670 nm or an absorbance microplate reader at ~647 nm. The kit comes with the following components:

• Amplite™ IR substrate• HRP and H2O2

• Assay buffer

Figure 5.23. HRP dose response was measured with the Amplite™ Colorimetric Peroxidase Assay Kit in a 96-well white wall/clear bottom plate. As low as 3 mU/mL of peroxidase can be detected.

Figure 5.24. HRP dose response was measured with the Amplite™ Fluorimetric Peroxidase Assay Kit in a 384-well black solid plate using a Gemini fluorescence microplate reader (Molecular Devices). As low as 1 mU/mL of peroxidase can be detected with 30 minutes incubation time (n=3).

Related Products









168737j1_cat.indd 48 2/4/11 11:54 AM




5

Peroxidase

Amplite™ Fluorimetric Peroxidase Assay Kit *Red Fluorescence*


11552 1 kit < - 15 oC

This kit uses our outstanding Amplite™ Red HRP substrate. Colorless and non-fluorescent Amplite™ Red generates an intense red fluores-cent product upon reaction with hydrogen peroxide in the presence of HRP. The increase in fluorescence intensity is proportional to HRP activity in the presence of H2O2.

We offer this quick HRP assay in a one-step, homogeneous, no wash assay system. This kit uses our fluorogenic Amplite™ Red HRP sub-strate to quantify peroxidase in solutions. It can be used for ELISAs, characterizing kinetics of enzyme reaction, and high throughput screenings. This Amplite™ Fluorimetric Peroxidase Assay Kit pro-vides an optimized “mix and read” assay protocol that is compatible with HTS liquid handling instruments. It can detect as low as 10 µU/mL of HRP. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format. Its signal can be easily read by either a fluorescence microplate reader at Ex/Em = ~540/590 nm or an absorbance microplate reader at ~576 nm. The kit has robust performance and comes with the following components:

• Amplite™ Red• HRP and H2O2

• Assay buffer

Amplite™ Luminometric Peroxidase Assay Kit


11559 1 kit < - 15 oC

Enhanced chemiluminescence is a common technique for a variety of detection assays in biology. A horseradish peroxidase enzyme (HRP) is tethered to the molecule of interest (usually through label-ing an immunoglobulin that specifically recognizes the molecule). This enzyme complex catalyzes the conversion of the enhanced chemiluminescent substrate into a sensitized reagent in the vicinity of the molecule of interest. The further oxidation of the substrate by hydrogen peroxide produces an excited molecule which emits light.

This kit uses our Amplite™ luminometric HRP substrate to quantify peroxidase in solutions. It provides an optimized “mix and read” assay protocol. Our Amplite™ Luminometric Peroxidase Assay Kit can detect as low as 100 µU/mL of HRP. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format. Its signal can be easily read by a luminescence microplate reader. The kit can be used for ELISAs, characterizing kinetics of enzyme reaction and high throughput screenings. The kit comes with the following components:

• HRP enzyme• Hydrogen peroxide• Assay buffer

Figure 5.25. HRP dose response was measured with the Amplite™ Fluorimetric Peroxidase Assay Kit in a 384-well black plate using a Gemini fluorescence microplate reader (Molecular Devices). As low as 10 µU/mL of peroxidase can be detected with 30 minutes incubation time (n=3).

Figure 5.26. HRP dose response was measured with the Amplite™ Luminometric Peroxidase Assay Kit in a 384-well black plate. As low as 100 µU/mL of peroxidase can be detected with 30 minutes incubation time (n=3).

Related Products









168737j1_cat.indd 49 2/4/11 11:54 AM



Related Products


50


5

HRP

Con

juga

te

Amplite™ Fluorimetric Goat Anti-Mouse IgG-HRP Conjugate ELISA Assay Kit *Red Fluorescence*


11540 1 kit < - 15 oC

Horseradish Peroxidase (HRP) is a small molecule (MW ~40 KD) that is widely used in a variety of biological detections. HRP conjugates are extensively used as secondary detection reagents in ELISAs, immuno-histochemical techniques as well as Northern, Southern and Western blot analyses. Due to its small size, HRP rarely causes any steric hindrance problem with the antibody/antigen complex formation. In addition, HRP conjugates are much more stable than other enzyme conjugates, making the HRP-based ELISA assays much more robust.

Our Amplite™ Fluorimetric ELISA Assay Kit contains all the essential components including our fluorogenic Amplite™ Red HRP substrate for ELISA detection. The kit provides an optimized assay protocol. It can detect as little as 0.4 ng/well of a monoclonal antibody. Its sig-nal can be easily read by either a fluorescence microplate reader at Ex/Em = ~540/590 nm or an absorbance microplate reader at ~576 nm. It has been used for the assays in which goat anti-mouse IgG is served as a secondary detection agent.

Amplite™ Fluorimetric Goat Anti-Rabbit IgG-HRP Conjugate ELISA Assay Kit *Red Fluorescence*


11541 1 kit < - 15 oC

This kit uses our outstanding Amplite™ Red HRP substrate. Colorless and non-fluorescent Amplite™ Red generates an intense red fluores-cent product upon reaction with hydrogen peroxide in the presence of HRP. The increase in fluorescence intensity is proportional to HRP activity in the presence of H2O2.

Our Amplite™ Fluorimetric ELISA Assay Kit can be used for the as-says in which goat anti-rabbit IgG is served as the secondary detec-tion agent. It provides an optimized assay protocol that is compat-ible with HTS liquid handling instruments. As little as 3 ng/well of a polyclonal antibody can be detected. Its signal can be easily read by either a fluorescence microplate reader at Ex/Em = ~540/590 nm or an absorbance microplate reader at ~576 nm. The kit is convenient to use and comes with the following components:

• Amplite™ Red substrate• Goat anti-rabbit IgG• Hydrogen peroxide• Assay buffer

Figure 5.27. Detection of mouse total IgG using the Amplite™ Fluorimetric ELISA Kit. Mouse IgG was diluted into 3 μg/mL and made 1 to 3 serial dilutions in 0.2 M sodium bicarbonate buffer, pH 9.4. 100 µL/well serial dilutions were coated into a 96-well solid black plate at 4 oC for overnight, and blocked with 3% milk in PBS and 0.02% Tween-20 at 4 oC for overnight. The wells were washed, and assayed using the reagents. 1 to 5000 dilutions of goat anti-mouse IgG, HRP conjugate were used. The reac-tions were incubated for 10 to 60 minutes and then measured for fluorescence at Ex/Em = 540/590 nm using Gemini fluorescence microplate reader (Molecular Devices). As low as 0.4 ng/well of mouse total IgG can be detected with 10 minutes incubation time (n=3).

Figure 5.28. Detection of total rabbit IgG using the Amplite™ Fluorimetric ELISA Kit. Rabbit IgG was diluted into 1 μg/mL and made 1 to 3 serial dilutions in 0.2 M sodium bicarbonate buffer at pH 9.4. 100 µL/well serial dilutions were coated into a 96-well black plate at 4 oC overnight, and blocked with 3% milk in PBS and 0.02% Tween-20 at 4 oC overnight. The wells were washed, and assayed using the reagents. 1 to 6000 dilutions of goat anti-rabbit IgG, HRP conjugate were used. The reactions were incubated for 15 to 60 minutes and then measured for fluorescence at Ex/Em = 540/590 nm. As low as 3 ng/well of total rabbit IgG can be detected with 30 minutes incubation time (n=3).

Related Products






11553 1 kit Amplite™ Fluorimetric Peroxidase Assay Kit *Near Infrared Fluorescence*



168737j1_cat.indd 50 2/4/11 11:54 AM




5

Proteasome 20S/Protease

Amplite™ Fluorimetric Proteasome 20S Activity Assay Kit *Green Fluorescence*


13456 1 kit < - 15 oC

The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis. The most common form of the proteasome in this pathway is the 26S proteasome, an ATP-depen-dent proteolytic complex, which contains one 20S (700-kDa) core particle structure and two 19S (700-kDa) regulatory caps. The 20S core contains three major proteolytic activities including chymo-trypsin-like, trypsin-like and caspase-like activities. It is responsible for the breakdown of the key proteins involved with apoptosis, DNA repair, endocytosis, and cell cycle control.

Our Amplite™ Fluorimetric Proteasome 20S Activity Assay Kit is a homogeneous fluorescent assay that measures the chymotrypsin-like protease activity associated with the proteasome complex in cultured cells. This kit uses LLVY-R110 as a fluorogenic indicator for proteasome activities. Cleavage of LLVY-R110 by proteasome gener-ates strongly green fluorescent R110 that is monitored fluorimetri-cally at 520-530 nm with excitation at 480-500 nm. The kit provides all the essential components with an optimized assay protocol. The assay is robust and can be readily adapted for high-throughput as-says to evaluate the proteasome activities or screen the inhibitors in cultured cells or in solution.

Amplite™ Universal Fluorimetric Protease Activity Assay Kit *Green Fluorescence*


13500 1 kit < - 15 oC

Protease assays are widely used for the investigation of protease inhibitors and detection of protease activities. Monitoring various protease activities has become a routine task for many biological laboratories. Some proteases have been identified as good drug development targets.

Our Amplite™ Universal Fluorimetric Protease Activity Assay Kit is an ideal choice to perform routine assays for the isolation of prote-ases, or for identifying the presence of contaminating proteases in protein samples. The kit uses a fluorescent casein conjugate which is proven to be a generic substrate for a broad spectrum of prote-ases (e.g. trypsin, chymotrypsin, thermolysin, proteinase K, protease XIV, and elastase). In the intact substrate, casein is heavily labeled with a green fluorescent dye, resulting in significant fluorescence quenching. Protease-catalyzed hydrolysis relieves its quenching effect, yielding brightly fluorescent dye-labeled short peptides. The increase in fluorescence intensity is directly proportional to prote-ase activity. The assay can be performed in a convenient 96-well or 384-well microtiter plate format and readily adapted to automation. Its signal can be easily read with a fluorescence microplate reader at Ex/Em = 490/525 nm using FITC filter set.

Figure 5.30. Trypsin protease activity was analyzed by Amplite™ Universal Fluorimetric Protease Activ-ity Assay kit. Protease substrate was incubated with 1 unit trypsin in the kit assay buffer. The control wells had protease substrate only (without trypsin). The fluorescence signal was measured starting from time 0 when trypsin was added. Samples were done in triplicates.

Figure 5.29. Detection of Proteasome Activity in Jurkat cells. Jurkat cells were seeded on the same day at 500,000 cells/90 µL/well in a 96-well black wall/clear bottom Costar plate. The cells were treated with or without 50 mM H2O2 for 30 minutes. The proteasome assay loading solution (100 µL/well) was added and incubated in a 5% CO2, 37 oC incubator for 3 hours. The fluorescence intensity was mea-sured at Ex/Em = 490/525 nm with Gemini fluorescent microplate reader (Molecular Devices).

Related Products


13401 5 mg Ac-DEVD-AFC

13402 5 mg Ac-DEVD-AMC


13455 1 mg (Ac-ANW)2 -R110

13431 1 mg (Ac-IETD)2-R110

13430 1 mg (Z-DEVD)2-R110





5

Pro

teas

e/Re

nin

Amplite™ Universal Fluorimetric Protease Activity Assay Kit *Red Fluorescence*


13501 1 kit < - 15 oC

Protease assays are widely used for the investigation of protease inhibitors and detection of protease activities. Monitoring various protease activities has become a routine task for many biological laboratories. Some proteases have been identified as good new drug development targets.

Our Amplite™ Universal Fluorimetric Protease Activity Assay Kit is an ideal choice to perform routine protease assays for the isolation of proteases, or for identifying the presence of contaminating proteas-es in samples. The kit uses a red fluorescent casein conjugate that is proven to be a generic substrate for a broad spectrum of proteases (e.g. trypsin, chymotrypsin, thermolysin, proteinase K, protease XIV, and elastase). In the intact substrate, casein is heavily labeled with a fluorescent dye, resulting in significant fluorescence quenching. Protease-catalyzed hydrolysis relieves its quenching effect, yield-ing brightly fluorescent dye-labeled short peptides. The increase in fluorescence intensity is directly proportional to protease activity. The assay can be performed in a convenient 96-well or 384-well microtiter plate format. Its signal can be easily read at Ex/Em = 540 /590 nm. This kit has been used for screening protease inhibitors in a HTS mode.

Amplite™ Fluorimetric Renin Assay Kit *Green Fluorescence*


13530 1 kit < - 15 oC

Renin is an enzyme that participates in the body's renin-angiotensin system (RAS) that mediates extracellular volume and arterial vaso-constriction. It regulates blood pressure and electrolyte homoeo-stasis. Angiotensin II constricts blood vessels leading to increased blood pressure. It also increases the secretion of ADH and aldoste-rone, and stimulates the hypothalamus to activate the thirst reflex. An over-active renin-angiotension system leads to vasoconstriction and retention of sodium and water. Renin has been identified to be an attractive target for the treatment of hypertension.

The Amplite™ Renin Assay Kit provides a convenient assay for high throughput screening of renin inhibitors and renin activity using our proprietary iFluor™ 488/TQ™ 520 fluorescence resonance energy transfer (FRET) peptide. In the FRET peptide, the fluorescence of iF-luor™488 is quenched by TQ™ 520. Upon cleavage into two separate fragments by renin, the fluorescence of iFluor™ 488 is recovered, and the fluorescent signal can be easily monitored by a fluorescence microplate reader at Ex/Em = 490/520 nm. This assay is about fifty fold more sensitive than an EDANS/DABCYL-based assay. With the Amplite™ Renin Assay Kit, we have detected as little as 1ng renin in a 100 µL reaction volume.

Figure 5.31. Trypsin protease activity was analyzed by using Amplite™ Universal Fluorimetric Protease Activity Assay Kit. Protease substrate was incubated with 3 units of trypsin. The fluorescence signal was measured starting from time 0 when trypsin was added. Samples were done in triplicate.

Related Products


13510 1 kit Amplite™ Universal Fluorimetric MMP Activity Assay Kit *Green Fluorescence*

13500 1 kit Amplite™ Universal Fluorimetric Protease Activity Assay Kit *Green Fluorescence*

13461 5 mg BOC-Val-Pro-Arg-AMC

13450 5 mg Gly-Pro-AMC

13455 1 mg (Ac-ANW)2-R110

13451 1 mg (Suc-LLVY)2-R110


Figure 5.32. Renin dose response was measured with Amplite™ Renin Assay Kit on a 96-well black solid plate using a Gemini fluorescence microplate reader (Molecular Devices). As low as 0.01ug/mL Renin was detected with 30 minutes incubation time (n=3).

168737j1_cat.indd 52 2/4/11 11:54 AM




Related Products


13610 100 nmol AMP-Fluorescein conjugate calibrator

13622 1 kit Amplite™ Fluorimetric Acid Sphingomyelinase Assay Kit *Red Fluorescence*

13602 0.5 umol FAM-cAMP PDE IV substrate *Green fluorescence*

13604 0.5 umol FAM-cGMP PDE V substrate *Green fluorescence*

13611 100 nmol GMP-Fluorescein conjugate calibrator

13603 0.5 umol TAMRA-cAMP PDE IV substrate *Red fluorescence*

13605 0.5 umol TAMRA-cGMP PDE V substrate *Red fluorescence*

5

Sphingomyelinase

Amplite™ Colorimetric Sphingomyelinase Assay Kit


13620 1 kit < - 15 oC

Sphingomyelinase (SMase) is an enzyme that is responsible for cleaving sphingomyelin (SM) to phosphocholine and ceramide. Activation of SMases in cells plays an important role in the cellu-lar responses. Five types of sphingomyelinase (SMase) have been identified based on their cation dependence and pH optima of action. They are lysosomal acid SMase, secreted zinc-dependent acid SMase, magnesium-dependent neutral SMase, magnesium-independent neutral SMase, and alkaline SMase. Among the five types, the lysosomal acidic SMase and the magnesium-dependent neutral SMase are considered major candidates for the production of ceramide in the cellular response to stress.

Our Amplite™ Colorimetric Sphingomyelinase Assay Kit provides a sensitive method for detecting neutral SMase activity or screening its inhibitors. The kit uses Amplite™ Blue as a colorimetric probe to indirectly quantify the phosphocholine produced from the hydro-lysis of sphingomyelin (SM) by sphingomyelinase (SMase). It can be used for measuring the SMase activity in blood, cell extracts or other solutions. The absorbance of light at 595 nm is proportional to the formation of phosphocholine, therefore to the SMase activity. The kit is an optimized “mix and read” assay that is compatible with HTS liquid handling instruments.

Amplite™ Fluorimetric Sphingomyelinase Assay Kit *Red Fluorescence*


13621 1 kit < - 15 oC

Five types of sphingomyelinase (SMase) have been identified based on their cation dependence and pH optima of action. They are lysosomal acid SMase, secreted zinc-dependent acid SMase, magnesium-dependent neutral SMase, magnesium-independent neutral SMase, and alkaline SMase. Among the five types, the lyso-somal acidic SMase and the magnesium-dependent neutral SMase are considered major candidates for the production of ceramide in the cellular response to stress.

Our Amplite™ Fluorimetric Sphingomyelinase Assay Kit provides the most sensitive method for detecting neutral SMase activity or screening its inhibitors. The kit uses Amplite™ Red as a fluorogenic probe to indirectly quantify the phosphocholine produced from the hydrolysis of sphingomyelin (SM) by sphingomyelinase (SMase). It can be used for measuring the SMase activity in blood, cell extracts or other solutions. The fluorescence intensity of Amplite™ Red is proportional to the formation of phosphocholine, therefore to the SMase activity. Amplite™ Red enables the assay readable either in fluorescence intensity or absorption mode. The kit is an optimized “mix and read” assay that can be used for real time monitoring of Smase activities. Our kit 13622 has been developed for monitoring acid SMase activity.

Figure 5.33. Sphingomyelinase dose response was measured in a 96-well white wall/clear bottom plate with Amplite™ Colorimetric Sphingomyelinase Assay Kit using a Spectrum Max microplate reader (Molecular Devices). As low as 0.08 mU/mL of sphingomyelinase can be detected with 4 hours incubation time (n=3).

Figure 5.34. Sphingomyelinase dose response was measured in a 96-well black solid plate with Amplite™ Fluorimetric Sphingomyelinase Assay Kit using a Gemini fluorescence microplate reader (Molecular Devices). As low as 0.15 mU/mL of sphingomyelinase can be detected with 60 minutes incubation time (n=3).




Related Products


11306 1 kit Amplite™ Fluorimetric Catalase Assay Kit *Red Fluorescence*

11300 1 kit Amplite™ Fluorimetric Glucose Oxidase Assay Kit *Red Fluorescence*

11302 1 kit Amplite™ Fluorimetric Glutamate Oxidase Assay Kit *Red Fluorescence*



11303 1 kit Amplite™ Fluorimetric Monoamine Oxidase Assay Kit *Red Fluorescence*

11301 1 kit Amplite™ Fluorimetric Myelopeoxidase Assay Kit *Red Fluorescence*

5

SOD

/Xan

thin

e O

xida

se

Amplite™ Colorimetric Superoxide Dismutase (SOD) Assay Kit


11305 1 kit < - 15 oC

Superoxide dismutases (SOD) are a class of enzymes that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. Superoxide is one of the main reactive oxygen species in cells. It is a substantial contributor of pathology associated with neurodegen-erative diseases, ischemia reperfusion injury, atherosclerosis and aging. SODs are an important antioxidant defense in nearly all cells exposed to superoxide radicals. In fact, mice lacking SOD1 develop a wide range of pathologies, including hepatocellular carcinoma, an acceleration of age-related muscle mass loss, an earlier incidence of cataracts and a reduced lifespan. Overexpression of SOD protects murine fibrosarcoma cells from apoptosis and promotes cell dif-ferentiation.

The Amplite™ Colorimetric Superoxide Dismutase (SOD) Assay Kit provides a quick and sensitive method for the measurement of SOD activity in solutions. In the assay, xanthine is converted to super-oxide radical ions, uric acid and hydrogen peroxide by xanthine oxidase (XO). Superoxide reacts with SOD Orange™ to generate a product that absorbs around 560 nm. SOD inhibits the reaction of SOD Orange™ with superoxide, thus reduces the absorption at 560 nm. The reduction in the abosoption of SOD Orange™ at 560 nm is proportional to SOD activity. The kit can be performed in a conve-nient 96-well or 384-well microtiter-plate format.

Amplite™ Fluorimetric Xanthine Oxidase Assay Kit *Red Fluorescence*


11304 1 kit < - 15 oC

Xanthine oxidase (XO) is an enzyme that catalyzes the oxidation of hypoxanthine to xanthine and can further catalyze the oxidation of xanthine to uric acid. It plays an important role in the catabolism of purines. Xanthine oxidase is normally found in liver and jejunum. During severe liver damage, xanthine oxidase is released into blood, so a blood assay for XO is a way to determine if liver damage has happened. Xanthinuria is a rare genetic disorder where the lack of xanthine oxidase leads to high concentration of xanthine in blood and can cause health problems such as renal failure.

The Amplite™ Xanthine Oxidase Assay Kit provides a quick and ultrasensitive method for the measurement of xanthine oxidase activities. It can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. In the assay, xanthine oxidase catalyzes the oxida-tion of purine bases, hypoxanthine or xanthine to uric acid and superoxide , which spontaneously degrades to hydrogen peroxide (H2O2). The kit uses our Amplite™ Red substrate which enables a dual recordable mode. The fluorescent signal can be easily read by either a fluorescence microplate reader at Ex/Em = 540/590 nm or an absorbance microplate reader at ~576 nm. With the Amplite™ Xanthine Oxidase Assay Kit, we have detected as little as 0.15 mU/mL xanthine oxidase in a 100 µL reaction volume.

Figure 5.35. SOD dose response was measured with Amplite™ Colorimetric Superoxide Dismutase (SOD) Assay Kit in a 96-well white wall/clear bottom plate with a Spectrum Max microplate reader (Molecular Devices). As low as 0.01 mU/mL SOD was detected with 30 minutes incubation time (n=3). .

Figure 5.36. Xanthine oxidase dose response was measured with Amplite™ Xanthine Oxidase Assay Kit on a 96-well black solid plate using a Gemini fluorescence microplate reader (Molecular Devices). As low as 0.15 mU/mL xanthine oxidase was detected with 30 minutes incubation time (n=3). .


Cell Labeling and Tracking

Cell Labeling and Tracking 6

168737j1_cat.indd 55 2/4/11 11:54 AM



Cell

Labe

ling

and

Trac

king

6

cell labeling and tracking products at-a-glance*

Cell CompartmentFluorescence Color

Blue Green Orange Red Near Infrared

F-Actin Labeling 22660 22661 22663 22664

Anti-Fading 20001 & 20003 20001 & 20003 20001 & 20003 20001 & 20003 20001 & 20003

Dead Cell Labeling 22500 & 22600 22501 & 22601 22502 & 22602 22603 22604

Live Cell Labeling 22606 & 22614 22607 & 22615 22616 22609

Live Cell Tracking 22620 22621 22622 22623

Lysosomal Staining 22655 22651 & 22656 22658

Mitochondrial Staining 22666 22673 & 22667 22668




www.aatbio.com Cell Labeling and Tracking

6

Anti-fading

FluoroQuest™ Anti-Fading Kit I *Optimized for Slide Imaging*


20001 1 kit < - 15 oC

The photon output of a dye represents the average number of cycles of excitation followed by fluorescence emission that the dye goes through before it is irreversibly photobleached. The average photon output is defined by the ratio of fluorescence quantum efficiency to photobleaching quantum efficiency. When exposed to excitation light, fluorescence intensity of dyes decreases due to their photooxidation or other photoreactions. It is ideal to have the maximal ratio of fluorescence quantum efficiency to photobleach-ing quantum efficiency. However, very few fluorescent organic dyes can completely resist photobleaching.

Frequently, when a section has been scanned repeatedly under strong excitation light, dyes could lose significant fluorescence signal before visual evaluation or photography can be accom-plished. For example, the photobleaching of fluoresceins (such as FITC-labeled antibodies) has become a major problem in fluores-cence microscopy. In severe cases (such as phycoprotein-labeled bioconjugates), a fluorescence image with high resolution cannot even be taken due to the extremely high photobleaching rate. The main purpose of FluoroQuest™ Anti-Fading Kits is to reduce the dye photobleaching rate, giving researchers longer observation time. Kit I contains 3 sampler components for different imaging experiments. The components are all premixed and ready-to-use solutions.

FluoroQuest™ Anti-Fading Kit II *Optimized for Plate Imaging*


20003 1 kit 2 - 6 oC

Light-induced damage to the fluorophores is prominent in the pres-ence of oxygen, most likely via the process mediated by singlet oxy-gen, a reactive oxygen species. In some cases, nonoxygen-mediated radical generation has been indicated as a source of photochemical destruction of fluorescent dyes.

Our FluoroQuest™ Anti-Fading kits have been developed to prevent the fading of fluorescent dyes. To be complementary to Kit I (the slide format), our FluoroQuest™ Anti-Fading Kit II is optimized for the microplate format. Unlike Kit I, Kit II provides only one formula-tion specifically optimized for FITC based experiments.

FluoroQuest™ Anti-Fading Kits are recommended for fixed cells and tissues that have been stained with fluorescent dyes or their biological conjugates (such as dye-antibody conjugates). Cells can be viewed immediately. If long term storage is desired, the reagent can be allowed to harden overnight. However, the staining patterns of FluoroQuest™ Anti-Fading Kits are sharper if viewed immediately. Our fluorescent reagents and assay kits have been extensively benchmarked for live cell analysis applications and are optimal for demanding cell analysis applications involving confocal microscopy, flow cytometry, microplate readers and HCS/HTS, where consis-tency and reproducibility are required.

Figure 6.1. U2OS cells in a 96-well Costar black wall/clear bottom plate were loaded with 1 µM calcein, AM for 1 hour and fixed with 4% formaldehyde for 30 minutes. Anti-fading reagents were added to the samples after removing all the media. The FITC signals were compared at 0 and after 30 seconds exposure using an Olympus fluorescence microscopy. The same exposure settings were used for all the images.

Related Products


22002 1 mg Calcein, AM

120 100 mg 5-FITC [FITC Isomer I; fluorescein-5-isothiocyanate] *Ultra Pure Grade*

21010 1 mg Fluo-3, AM

21080 10 mg Quest Fluo-8™, AM

392 5 mg 6-ROX, SE [6-Carboxy-X-rhodamine, succinimidyl ester] *Single isomer*

480 1 mg Sulforhodamine 101 sulfonyl chloride

372 5 mg 5-TAMRA, SE [5-Carboxytetramethylrhodamine, succinimidyl ester] *Single isomer*

168737j1_cat.indd 57 2/4/11 11:54 AM



Cell Labeling and Tracking www.aatbio.com

6

Dea

d Ce

ll La

belin

g

Cell Explorer™ Fixable Dead Cell Labeling Kit *Blue Fluorescence*


22600 1 kit < - 15 oC

Our Cell Explorer™ fluorescence labeling kits are a set of tools used to label cells for fluorescence microscopic and flow cytometric investigations of cellular functions. The effective labeling of cells provides a powerful method for studying cellular events in a spatial and temporal context.

The proprietary blue fluorescent dye used in the kit, which becomes more fluorescent upon binding to cellular components, has the spectral properties almost identical to those of AMCA. The fluores-cence signal of the blue fluorescent dye is pH-independent and quite photostable. The dead cell stain used in the kit is well excited at ~350 nm, and emits intense blue fluorescence at ~450 nm. Compared to AMCA, the dye used in the kit has much better water solubility, making the kit easier to use. This Cell Explorer™ Fixable Dead Cell Labeling Kit provides all the essential components with an optimized fixable dead cell-labeling protocol, which is straight-forward with minimal hands on time. This kit can be used with ei-ther a flow cytometer or a fluorescence microscope equipped with the standard DAPI or AMCA filter set (Ex/Em = 350/450 nm). It is an excellent tool for preserving fluorescent images of particular cells, and can also be used for fluorescence microscope demonstrations.

Cell Explorer™ Fixable Dead Cell Labeling Kit *Blue Fluorescence with 405 nm Excitation*


22500 1 kit < - 15 oC

Cell Explorer™ Fixable Dead Cell Labeling kits are a set of tools to label cells for flow cytometry and fluorescence microscopic investi-gations of cell functions. This particular kit is designed to uniformly label dead mammalian cells in blue fluorescence for long term microscopic examination and flow cytometry analysis.

This kit uses a proprietary blue fluorescent dye that is more fluo-rescent upon binding to cellular components. The fluorescent dye has the spectral properties almost identical to those of Pacific Blue® (Pacific Blue is the trademark of Invitrogen). The dead cell stain used in the kit emits intense blue fluorescence at ~450 nm when it is well excited with the Violet Laser at 405 nm. The large Strokes Shift of the stain used in the kit minimizes its staining background.

The kit provides all the essential components with an optimized cell-labeling protocol. It is an excellent tool for preserving fluo-rescent images of particular cells. The kit is optimized for a flow cytometry equipped with Violet Laser and can also be used with a fluorescence microscope using the Pacific Blue filter set (Ex/Em = 405/450 nm). It is widely used in multicolor flow cytometric analysis of cells in combination with fluorescent antibody conjugates.

Figure 6.2. Image of Hela cells fixed with formaldehyde and stained with Cell Explorer™ Fixable Dead Cell Labeling Kit (22600) in a 96-well Costar black wall/clear bottom plate.

Related Products


22003 1 mg Calcein, AM *UltraPure grade*

22009 1 mg Calcein Orange™

22010 1 mg Calcein Red™

22012 1 mg CytoCalcein™ Violet 450 *Excited at 405 nm*


22023 1 mg VFSE™ 450

22024 1 mg VFSE™ 500


168737j1_cat.indd 58 2/4/11 11:54 AM



Related Products


59


6

Dead Cell Labeling

Cell Explorer™ Fixable Dead Cell Labeling Kit *Deep Red Fluorescence*


22604 1 kit < - 15 oC

This particular Cell Explorer™ Fixable Dead Cell Labeling Kit is designed to uniformly label dead mammalian cells in deep red fluorescence. The stain used in the kit is well excited by the 633 nm He-Ne Laser that is installed on most flow cytometers. The labeling is optimized for the flow cytomtetric analysis of cells in multicolor applications.

The proprietary deep red fluorescent dye used in the kit becomes more fluorescent upon binding to cellular components. It is quite photostable, thus the images can be repeatedly examined. With the spectral properties almost identical to those of Cy5® or Alexa Fluor® 647 (Cy5® or Alexa Fluor® 647 are the trademarks of GE Healthcare and Invitrogen respectively), the dye can be conveniently used for fluorescence microscope and flow cytometry demonstrations with the common fluorescence instruments equipped with the light sources and a Cy5® or Alexa Fluor® 647 filter set (Ex/Em = 650/670 nm). This Cell Explorer™ Fixable Dead Cell Labeling Kit provides all the essential components with an optimized cell labeling proto-col. It is an effective tool for preserving the fluorescent images of particular cells.

Cell Explorer™ Fixable Dead Cell Labeling Kit *Green Fluorescence*


22601 1 kit < - 15 oC

Cell Explorer™ fluorescence Labeling kits are a set of tools to label cells for fluorescence microscopic and flow cytometric investiga-tions of cellular functions. This particular kit is designed to uniformly label dead mammalian cells in green fluorescence for long term microscopic examination.

The kit provides all the essential components with an optimized cell-labeling protocol. The proprietary green fluorescent dye used in the kit becomes more fluorescent upon binding to dead cellular components. When well excited at ~490 nm, the dead cell stain emits intense green fluorescence at ~520 nm. The fluorescent dye used in the kit has the spectral properties almost identical to those of FITC, but shows much better photostability than FITC. The kit can be readily used with almost every fluorescence instrument often equipped with a 488 nm Argon Laser. The dye used in the kit has high photostability. It can also be used with either a flow cytometer or a fluorescence microscope equipped with the standard FITC filter (Ex/Em = 490/520 nm). It is also an excellent tool for preserving fluorescent images of particular cells, and good for fluorescence microscope demonstrations.

Figure 6.4. Image of HeLa cells fixed with formaldehyde and stained with Cell Explorer™ Fixable Dead Cell Labeling Kit (22604) in a 96-well Costar black wall/clear bottom plate.

Figure 6.5. Image of CHO cells fixed with formaldehyde and stained with Cell Explorer™ Fixable Dead Cell Labeling Kit (22601) in a 96-well Costar black wall/clear bottom plate.

Related Products







22023 1 mg VFSE™ 450

22024 1 mg VFSE™ 500

168737j1_cat.indd 59 2/4/11 11:54 AM



Related Products


60


6

Dea

d Ce

ll La

belin

g

Cell Explorer™ Fixable Dead Cell Labeling Kit *Green Fluorescence with 405 nm Excitation*


22501 1 kit < - 15 oC

This particular Cell Explorer™ Fixable Dead Cell Labeling Kit is de-signed to uniformly label dead mammalian cells in green fluores-cence. The stain used in the kit is well excited by the 405 nm Violet Laser with emission around 510 nm. The labeling is optimized for the flow cytomtetric analysis of cellular functions in multicolor ap-plications.

The proprietary green fluorescent dye used in the kit is character-ized by a large Stokes Shift at Ex/Em = ~420/510 nm and becomes more fluorescent upon binding to dead cellular components. When excited with the Violet Laser at 405 nm, the dye emits intense green fluorescence at ~510 nm. The kit is optimized for a flow cytometer equipped with a Violet Laser and particularly suitable for multicolor flow cytometric analysis of cells. It may be also served as an excel-lent tool for fluorescence microscope demonstrations and the pres-ervation of fluorescent images of particular cells. Our Cell Explorer™ Fixable Dead Cell Labeling Kit provides an optimized cell-labeling protocol and comes with all the essential components:

• StainIt™ solution• Assay buffer

Cell Explorer™ Fixable Dead Cell Labeling Kit *Orange Fluorescence*


22602 1 kit < - 15 oC

Cell Explorer™ Fixable Dead Cell Labeling kits are a set of tools for labeling cells for fluorescence microscopic and flow cytometric investigations of cell functions. This particular kit is designed to uniformly label dead mammalian cells in orange fluorescence for microscopic examination and flow cytometry analysis.

The proprietary orange dye used in the kit becomes more fluores-cent upon binding to cellular components. It is quite photostable, thus the images can be repeatedly examined. With the spectral properties almost identical to those of Cy3® or Alexa Fluor® 555 (Cy3® or Alexa Fluor® 555 are the trademarks of GE Healthcare and Invitrogen respectively), it can be conveniently used with the common fluorescence instruments equipped with the light sources and filters for Cy3® or Alexa Fluor® 555. This Cell Explorer™ Fixable Dead Cell Labeling Kit provides all the essential components with an optimized cell labeling protocol. It is an effective tool for labeling dead cells and preserving the fluorescent images. The kit can be used for both flow cytometry analysis and fluorescence microscopic investigations of cell functions by using a Cy3® or Alexa Fluor® 555 filter set (Ex/Em = 540/590 nm).


Related Products


22500 1 kit Cell Explorer™ Fixable Dead Cell Labeling Kit *Blue Fluorescence with 405 nm Excitation*

22604 1 kit Cell Explorer™ Fixable Dead Cell Labeling Kit *Deep Red Fluorescence*

22501 1 kit Cell Explorer™ Fixable Dead Cell Labeling Kit *Green Fluorescence with 405 nm Excitation*

22502 1 kit Cell Explorer™ Fixable Dead Cell Labeling Kit *Orange Fluorescence with 405 nm Excitation*

22603 1 kit Cell Explorer™ Fixable Dead Cell Labeling Kit *Red Fluorescence*

22023 1 mg VFSE™ 450

22024 1 mg VFSE™ 500


168737j1_cat.indd 60 2/4/11 11:54 AM



Related Products


61


6

Dead Cell Labeling

Cell Explorer™ Fixable Dead Cell Labeling Kit *Orange Fluorescence with 405 nm Excitation*


22502 1 kit < - 15 oC

This particular Cell Explorer™ Fixable Dead Cell Labeling Kit is designed to uniformly label dead mammalian cells in orange fluorescence. The stain used in the kit is well excited by the 405 nm Violet Laser with emission around 560 nm. The labeling is optimized for the flow cytomtetric analysis of cellular functions in multicolor applications.

The proprietary orange fluorescent dye used in the kit has the spec-tral properties similar to those of Pacific Orange® (Pacific Orange® is the trademark of Invitrogen). It becomes more fluorescent upon binding to cellular components. When well excited with the Violet Laser at 405 nm, the dye emits intense orange fluorescence at ~550 nm. This Cell Explorer™ Fixable Dead Cell Labeling Kit provides all the essential components with an enhanced cell-labeling protocol. Optimized for a flow cytometer equipped with a Viloet Laser, it can also be used with a fluorescence microscope equipped with a proper filter set. The kit is an excellent tool to preserve fluorescent images of particular cells for fluorescence microscope and flow cytometry demonstrations by using the Pacific Orange® filter (Ex/Em = 405/560 nm).


Cell Explorer™ Fixable Dead Cell Labeling Kit *Red Fluorescence*


22603 1 kit < - 15 oC

Our Cell Explorer™ fluorescence imaging kits are a set of cell-labeling tools for fluorescence microscopic and flow cytometric investigations of cellular functions. The effective labeling of cells provides a powerful method for studying cellular events in a spatial and temporal context.

This particular kit is designed to uniformly label dead mammalian cells in red fluorescence for microscopic and flow cytometric exami-nation. The proprietary red dye used in the kit has the spectral prop-erties similar to those of Texas Red® (Texas Red® is the trademark of Invitrogen). It becomes more fluorescent upon binding to cellular components. When well excited at ~580 nm, the dye emits intense red fluorescence at ~610 nm. The red stain used in the kit has high photostability. The kit provides all the essential components with an optimized cell-labeling protocol. As an excellent tool to preserve fluorescent images of particular cells, it can be used with a flow cytometer or fluorescence microscope using the Texas Red® filter set (Ex/Em = 580/610 nm) installed with most of imaging instruments and flow cytometers.


Related Products


22500 1 kit Cell Explorer™ Fixable Dead Cell Labeling Kit *Blue Fluorescence with 405 nm Excitation*


22501 1 kit Cell Explorer™ Fixable Dead Cell Labeling Kit *Green Fluorescence with 405 nm Excitation*

22502 1 kit Cell Explorer™ Fixable Dead Cell Labeling Kit *Orange Fluorescence with 405 nm Excitation*


22023 1 mg VFSE™ 450

22024 1 mg VFSE™ 500

168737j1_cat.indd 61 2/4/11 11:54 AM



Related Products


62


6

Live

Cel

l Lab

elin

g

Cell Explorer™ Live Cell Labeling Kit *Blue Fluorescence*


22606 1 kit < - 15 oC

Our Cell Explorer™ fluorescence labeling kits are a set of tools which can be used to label cells for fluorescence microscopic and flow cytometric investigations of cellular functions. The effective labeling of cells provides a powerful method for studying cellular events in a spatial and temporal context.

This particular kit is designed to uniformly label live cells in blue fluorescence with a proprietary dye whose fluorescence is strongly enhanced upon entering into live cells. The dye is a hydrophobic compound that easily permeates intact live cells. The hydrolysis of the weakly fluorescent substrate by intracellular esterases generates a strongly fluorescent hydrophilic product that is well-retained in the cell cytoplasm. Cells grown on black wall/clear bottom plates or slides can be stained and quantified in less than two hours. This Cell Explorer™ fluorescence labeling kit can be readily adapted for a wide variety of fluorescence platforms such as microplate assays, flow cytometry and fluorescence microscope. It is useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. The kit provides all the essential components and can be used for both suspension or adherent cells.

Cell Explorer™ Live Cell Labeling Kit*Blue Fluorescence with 405 nm Excitation*


22614 1 kit < - 15 oC

The proprietary blue fluorescent dye used in the kit is a hydro-phobic compound. It can easily permeate intact live cells and get enhanced fluorescence upon entering into live cells. The hydrolysis of the weakly fluorescent substrate by intracellular esterases gener-ates a strongly fluorescent hydrophilic product that is well-retained in the cell cytoplasm. The blue fluorescence dye has the spectral properties similar to those of Pacific Blue® at Ex/Em = ~400/450 nm (Pacific Blue® is the trademark of Invitrogen). When excited with the Violet Laser at 405 nm, the dye emits intense blue fluorescence at ~450 nm. The kit is optimized to be used with a flow cytometry equipped with a Violet Laser and particularly suitable for multicolor flow cytometric analysis of cells. This kit can also be used with a fluorescence microscope with a Pacific Blue® filter set.

This Cell Explorer™ Live Cell Labeling Kit provides all the essential components with an optimized cell-labeling protocol (Ex/Em = 405/450 nm). It is an effective tool of labeling cells for flow cytomet-ric and fluorescence microscopic investigations of cellular functions. This kit is useful for a variety of studies, including cell adhesion, che-motaxis, multidrug resistance, cell viability, apoptosis and cytotoxic-ity. It is suitable for proliferating and non-proliferating cells.

Figure 6.10. Image of HeLa cells stained with Cell Explorer™ Live Cell Labeling Kit (22606) in a 96-well Costar black wall/clear bottom plate.


Related Products







22023 1 mg VFSE™ 450

22024 1 mg VFSE™ 500

168737j1_cat.indd 62 2/4/11 11:54 AM



Related Products


63


6

Live Cell Labeling

Cell Explorer™ Live Cell Labeling Kit *Green Fluorescence*


22607 1 kit < - 15 oC

Our Cell Explorer™ Live Cell Labeling Kits are a set of tools which can be used to label cells for fluorescence microscopic and flow cytometric investigations of cellular functions. The effective labeling of cells provides a powerful method for studying cellular events in a spatial and temporal context.

This particular kit is designed to uniformly label live cells in green fluorescence. The kit uses non-fluorescent Calcein Green™ that becomes strongly fluorescent upon entering into live cells. Calcein Green™ is a hydrophobic compound that easily permeates intact live cells. The hydrolysis of the non-fluorescent Calcein Green™ by intracellular esterases generates the strongly fluorescent hydrophil-ic calcein that is well-retained in the cell cytoplasm. Cells grown on black wall/clear bottom plates or slides can be stained and quanti-fied in less than two hours. This Cell Explorer™ Live Cell labeling kit can be readily adapted for a wide variety of fluorescence platforms such as microplate assays, flow cytometry and fluorescence mi-croscope. It is useful for a variety of studies, including cell adhe-sion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. The kit provides all the essential components with an optimized cell-labeling protocol (Ex/Em=490/525), and can be used for both proliferating and non-proliferating cells (either suspension or adherent cells).

Cell Explorer™ Live Cell Labeling Kit *Green Fluorescence with 405 nm Excitation*


22615 1 kit < - 15 oC

The proprietary green fluorescent dye used in the kit is a hydro-phobic compound. It can easily permeate intact live cells and get enhanced fluorescence upon entering into live cells. The hydrolysis of the weakly fluorescent substrate by intracellular esterases gener-ates a strongly fluorescent hydrophilic product that is well-retained in the cell cytoplasm. The green fluorescence dye has the spectral properties of large Stokes Shift at Ex/Em = ~410 nm/510 nm. When well excited with the Violet Laser at 405 nm, it emits intense green fluorescence at ~510 nm. The kit is optimized to be used with a flow cytometry equipped with a Violet Laser and particularly suitable for multicolor flow cytometric analysis of cells. It can also be used with a fluorescence microscope with a customized filter set.

This Cell Explorer™ Live Cell Labeling Kit provides all the essential components with an optimized cell-labeling protocol (Ex/Em = 405/510 nm). It is an excellent tool to label cells for fluorescence microscopic and flow cytometric investigations of cellular func-tions. The effective labeling of cells provides a powerful method for studying cellular events in a spatial and temporal context. This kit is useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. It is suitable for proliferating and non-proliferating cells.

Figure 6.12. Image of HeLa cells were stained with Cell Explorer™ Live Cell Labeling Kit (22607) in a 96-well Costar black wall/clear bottom plate.

Figure 6.13. Image of HeLa cells were stained with Cell Explorer™ Live Cell Labeling Kit (22615) in a 96-well Costar black wall/clear bottom plate.

Related Products







22023 1 mg VFSE™ 450

22024 1 mg VFSE™ 500

168737j1_cat.indd 63 2/4/11 11:54 AM



Related Products


64


6

Live

Cel

l Lab

elin

g

Cell Explorer™ Live Cell Labeling Kit *Orange Fluorescence with 405 nm Excitation*


22616 1 kit < - 15 oC

The proprietary orange fluorescent dye used in the kit is a hydro-phobic compound that easily permeates intact live cells. It gets enhanced fluorescence upon entering into live cells. The hydrolysis of the weakly fluorescent substrate by intracellular esterases gener-ates a strongly fluorescent hydrophilic product that is well-retained in the cell cytoplasm. The dye has the spectral properties of large Stokes Shift at Ex/Em = ~420 nm/560 nm. When well excited with the Violet Laser at 405 nm, it emits intense orange fluorescence at ~560 nm. It is optimized to be used with a flow cytometry equipped with a Violet Laser and particularly suitable for multicolor flow cyto-metric analysis of cells. This kit can also be used with a fluorescence microscope with a Pacific Orange® filter set (Pacific Orange® is the trademark of Invitrogen).

This Cell Explorer™ Live Cell labeling Kit is an excellent tool of label-ing cells for fluorescence microscopic and flow cytometric investiga-tions of cellular functions. The effective labeling of cells provides a powerful method for studying cellular events in a spatial and tem-poral context. This kit is useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. It is suitable for proliferating and non-proliferating cells, and can be used for both suspension and adherent cells.

Cell Explorer™ Live Cell Labeling Kit *Red Fluorescence*


22609 1 kit < - 15 oC


This particular kit is designed to uniformly label live cells in red fluorescence with a proprietary non-fluorescent dye that becomes strongly fluorescence upon entering into live cells. The dye is a hydrophobic compound that easily permeates intact live cells. The hydrolysis of the non-fluorescent substrate by intracellular esterases generates a strongly red fluorescent hydrophilic product that is well-retained in the cell cytoplasm. Cells grown on slides or black wall/clear bottom plates can be stained and quantified in less than two hours. The assay is more robust than the tetrazolium salt or Alarmar Blue™-based ones. It can be readily adapted for many differ-ent types of fluorescence platforms such as microplate assays, flow cytometry and fluorescence microscope with Ex/Em = 646/660 nm. And it is useful in a variety of studies, including cell adhesion, che-motaxis, multidrug resistance, cell viability, apoptosis and cytotoxic-ity. The kit provides all the essential components and can be used for both proliferating and non-proliferating cells.



Related Products







22023 1 mg VFSE™ 450

22024 1 mg VFSE™ 500

168737j1_cat.indd 64 2/4/11 11:54 AM



Related Products


65


6

Cell Tracking

Cell Explorer™ Live Cell Tracking Kit *Blue Fluorescence*


22620 1 kit < - 15 oC

Our Cell Explorer™ Live Cell labeling kits are a set of tools used to la-bel cells for fluorescence microscopic investigations of cellular func-tions. The effective labeling of cells provides a powerful method for studying cellular events in a spatial and temporal context.

This particular kit is designed to uniformly label live cells in blue fluorescence for the studies that require the fluorescent tag mol-ecules retained inside cells for a relatively longer time. The kit uses a non-fluorescent dye that carries a cell-retaining moiety. The dye becomes strongly fluorescent upon entering into live cells, and is trapped inside cells to give stable fluorescence signals. The dye is a hydrophobic compound that easily permeates intact live cells. The labeling process is robust, requiring minimal hands-on time. This Cell Explorer™ Live Cell labeling kit can be readily adapted for many different types of fluorescence platforms such as microplate assays, flow cytometry and fluorescence microscope. It is useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. The kit provides all the essential components with an optimized cell-labeling pro-tocol, and can be used for both proliferating and non-proliferating cells (either suspension or adherent cells).

Cell Explorer™ Live Cell Tracking Kit *Green Fluorescence*


22621 1 kit < - 15 oC

This particular kit is designed to uniformly label live cells in green fluorescence for the studies that require the fluorescent tag mol-ecules retained inside cells for a relatively longer time. The kit uses a non-fluorescent dye that carries a cell-retaining moiety. The dye becomes strongly fluorescent upon entering into live cells, and trapped inside cells to give a stable fluorescence signal. The labeling process is robust and convenient, requiring minimal hands-on time.

As kit 22620, this kit can be readily adapted for many different types of fluorescence platforms such as microplate assays, immunocyto-chemistry and flow cytometry. It is useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. The green fluorescent indica-tor used in the kit has Ex/Em = 490/520 nm, compatible with the FITC filter set that is installed with almost every major fluorescence instrument. The kit provides all the essential components with an optimized cell-labeling protocol, and can be used for both prolif-erating and non-proliferating cells (either suspension or adherent cells). Due to its minimal cytotoxicity of the dye, this kit can be used for monitoring the interaction of a drug compound with its cellular targets in live cells.

Figure 6.16. Image of HeLa cells stained with Cell Explorer™ Live Cell Tracking Kit (22620) in a 96-well Costar black wall/clear bottom plate.


Related Products







22023 1 mg VFSE™ 450

22024 1 mg VFSE™ 500

168737j1_cat.indd 65 2/4/11 11:54 AM



Related Products


66


6

Cell

Trac

king

Cell Explorer™ Live Cell Tracking Kit *Orange Fluorescence*


22622 1 kit < - 15 oC

Our Cell Explorer™ Live Cell Tracking Kit uses a proprietary orange fluorescent dye that gets enhanced fluorescence upon entering into live cells. The dye is a hydrophobic compound that easily permeates intact live cells. The hydrolysis of the weakly fluorescent substrate by intracellular esterases generates a strongly fluorescent hydrophilic product that is well-retained in the cell cytoplasm. The fluorescent dye used in the kit has the spectral properties of Ex/Em = ~530/550 nm. The tracking dye has good photostability with robust imag-ing performance. The kit is particularly suitable for multicolor flow cytometric analysis of cells. It can also be used with a fluorescence microscope equipped with a TRITC filter set.

This kit provides an effective tool of labeling cells for flow cytomet-ric and fluorescence microscopic investigations of cellular functions. The effective labeling of cells offers a powerful method for studying cellular events in a spatial and temporal context. This kit is useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. It is suitable for proliferating and non-proliferating cells, and can be used for both suspension and adherent cells. The kit provides all the essential components with an optimized cell-labeling protocol.

Cell Explorer™ Live Cell Tracking Kit *Red Fluorescence*


22623 1 kit < - 15 oC


This particular kit is designed to uniformly label live cells in red fluorescence for the studies that require the fluorescent tag mol-ecules retained inside cells for a relatively longer time. The kit uses a non-fluorescent dye that carries a cell-retaining moiety. The dye is a hydrophobic compound that easily permeates intact live cells. It becomes strongly fluorescent upon entering into live cells, and trapped inside to give a stable fluorescence signal. The labeling pro-cess is robust and convenient, requiring minimal hands-on time. The kit can be readily adapted for many different types of fluorescence platforms such as microplate assays, flow cytometry and fluores-cence microscope (Ex/Em = 575/600 nm). It is useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. The kit provides all the essential components with an optimized cell-labeling protocol, and can be used for both proliferating and non-proliferating cells.


Related Products







22023 1 mg VFSE™ 450

22024 1 mg VFSE™ 500

Figure 6.18. Image of Hela cells stained with Cell Explorer™ Live Cell Tracking Kit (22622) in a 96-well Costar black wall/clear bottom plate.

168737j1_cat.indd 66 2/4/11 11:54 AM



Related Products


67


6

F-Actin Labeling

Cell Navigator™ F-Actin Labeling Kit *Blue Fluorescence*


22660 1 kit < - 15 oC

Actin is a globular, roughly 42-kDa protein found in almost all eukaryotic cells. It is also one of the most highly-conserved proteins, differing by no more than 20% in species as diverse as algae and humans. Actin is the monomeric subunit of two types of filaments in cells: microfilaments, one of the three major components of the cytoskeleton, and thin filaments, part of the contractile apparatus in muscle cells. Thus, actin participates in many important cellular processes including muscle contraction, cell motility, cell division and cytokinesis, vesicle and organelle movement, cell signaling, as well as the establishment and maintenance of cell junctions and cell shape.

This particular kit is designed to label F-actins of fixed cells in blue fluorescence. The kit uses a blue fluorescent phalloidin conjugate that is selectively bound to F-actins. The phalloidin conjugate has Ex/Em = 350/450 nm, compatible with DAPI filter set that comes with most of fluorescence microscopes. It is a high-affinity probe for labeling, identifying and quantitating F-actins in formaldehyde-fixed and permeabilized tissue sections, cell cultures or cell-free experiments. The kit provides all the essential components with an optimized labeling protocol.

Cell Navigator™ F-Actin Labeling Kit *Green Fluorescence*


22661 1 kit < - 15 oC

Our Cell Navigator™ fluorescence imaging kits are a set of fluores-cence imaging tools for labeling sub-cellular organelles such as membranes, lysosomes, mitochondria, nuclei, etc. The selective labeling of live cell compartments provides a powerful method for studying cellular events in a spatial and temporal context.

This particular kit is designed to label F-actins in fixed cells with green fluorescence. The kit uses a green fluorescent phalloidin con-jugate that is selectively bound to F-actins. This green fluorescent phalloidin conjugate is a high-affinity probe for F-actins. When used at nanomolar concentrations, phallotoxins are convenient probes for labeling, identifying and quantitating F-actins in formaldehyde-fixed and permeabilized tissue sections, cell cultures or cell-free experiments. The kit provides all the essential components with an optimized staining protocol, which is robust requiring minimal hands-on time. It is an excellent tool for preserving the fluorescent images of particular cells. The phalloidin conjugate has spectral properties similar to those of FITC (Ex/Em = 500/520 nm). It can also be used for fluorescence microscope demonstrations using FITC filter (Ex/Em = 490/525 nm).

A B

Figure 6.20. Images of fixed CPA cells stained with Cell Navigator™ F-Actin Labeling Kit (22660) in a 96-well Costar black wall/clear bottom plate A: Label the cells with 1X iFluor™ 350-Phalloidin for 30 min only. B: Treat the cells with phalloidin for 10 min, then stain them with 1X iFluor™ 350-Phalloidin for 30 min.

A B

Figure 6.21. Image of fixed CPA cells stained with Cell Navigator™ F-Actin Labeling Kit (22661) in a 96-well Costar black wall/clear bottom plate A: Label the cells with 1X iFluor™ 488-Phalloidin for 30 minutes only. B: Treat the cells with phalloidin for 10 minutes, then stain them with 1X iFluor™ 488-Phalloidin for 30 minutes.

Related Products


22600 1 kit Cell Explorer™ Fixable Dead Cell Labeling Kit *Blue Fluorescence*

22601 1 kit Cell Explorer™ Fixable Dead Cell Labeling Kit *Green Fluorescence*

22602 1 kit Cell Explorer™ Fixable Dead Cell Labeling Kit *Orange Fluorescence*


22606 1 kit Cell Explorer™ Live Cell Labeling Kit *Blue Fluorescence*

22607 1 kit Cell Explorer™ Live Cell Labeling Kit *Green Fluorescence*

22609 1 kit Cell Explorer™ Live Cell Labeling Kit *Red Fluorescence*

168737j1_cat.indd 67 2/4/11 11:54 AM



Related Products


68


6

F-Ac

tin L

abel

ing

Cell Navigator™ F-Actin Labeling Kit *Orange Fluorescence*


22663 1 kit < - 15 oC

Actin is a globular, roughly 42-kDa protein found in almost all eukaryotic cells. It is also one of the most highly-conserved proteins, differing by no more than 20% in species as diverse as algae and humans. Actin is the monomeric subunit of two types of filaments in cells: microfilaments, one of the three major components of the cytoskeleton, and thin filaments, part of the contractile apparatus in muscle cells. Actin participates in many important cellular processes including muscle contraction, cell motility, cell division and cytoki-nesis, vesicle and organelle movement, cell signaling, as well as the establishment of cell junctions and cell shape.

This particular kit is designed to label F-actins of fixed cells in orange fluorescence. The kit uses an orange fluorescent phalloidin conjugate that is selectively bound to F-actins. The orange fluores-cent phalloidin conjugate has Ex/Em = 550/575 nm. The conjugate also has good photostability for fluorescence imaging. It is a high-affinity probe for labeling, identifying and quantitating F-actins in formaldehyde-fixed and permeabilized tissue sections, cell cultures or cell-free experiments. The kit provides all the essential compo-nents with an optimized labeling protocol.

Cell Navigator™ F-Actin Labeling Kit*Red Fluorescence*


22664 1 kit < - 15 oC

Our Cell Navigator™ fluorescence imaging kits are a set of fluores-cence tools for labeling sub-cellular organelles such as membranes, lysosomes, mitochondria and nuclei, etc. The selective labeling of live cell compartments provides a powerful method for studying cellular events in a spatial and temporal context.

This particular kit is designed to label F-actins of fixed cells in red fluorescence. The red fluorescent phalloidin conjugate, which is selectively bound to F-actins, is a high-affinity probe for F-actins. The red fluorescent phalloidin conjugate has Ex/Em = 588/605 nm, spectrally identical to the widely used Texas Red® conjugates. Used at nanomolar concentrations, phallotoxins can be conveniently used to label, identify and quantitate F-actins in formaldehyde-fixed and permeabilized tissue sections, cell cultures or cell-free experi-ments. The red fluorescent phalloidin conjugate has good thermal and photo stability. The kit provides all the essential components with an optimized labeling protocol. It is an excellent tool for pre-serving fluorescent images of particular cells, and can also be used for fluorescence microscope demonstrations using a Texas Red® filter (Texas Red is the trademark of InVitrogen).

A B

Figure 6.22. Images of fixed CPA cells stained with Cell Navigator™ F-Actin Labeling Kit (22663) in a 96-well Costar black wall/clear bottom plate. A: Label the cells with 1X iFluor™ 546-Phalloidin for 30 minutes only. B: Treat the cells with phalloidin for 10 minutes, then stain them with 1X iFluor™ 546-Phalloidin for 30 minutes.

Related Products


22600 1 kit Cell Explorer™ Fixable Dead Cell Labeling Kit *Blue Fluorescence*

22601 1 kit Cell Explorer™ Fixable Dead Cell Labeling Kit *Green Fluorescence*



22606 1 kit Cell Explorer™ Live Cell Labeling Kit *Blue Fluorescence*

22607 1 kit Cell Explorer™ Live Cell Labeling Kit *Green Fluorescence*

22609 1 kit Cell Explorer™ Live Cell Labeling Kit *Red Fluorescence*

A B

Figure 6.23. Images of fixed CPA cells stained with Cell Navigator™ F-Actin Labeling Kit (22664) in a 96-well Costar black wall/clear bottom plate. A: Label the cells with 1X iFluor™ 594-Phalloidin for 30 min only. B: Treat the cells with phalloidin for 10 min, then stain them with 1X iFluor™594-Phalloidin for 30 min.

168737j1_cat.indd 68 2/4/11 11:54 AM



Related Products


69


6

Lysosomal Staining

Cell Navigator™ Lysosomal Staining Kit *Blue Fluorescence*


22655 1 kit < - 15 oC


This particular kit is designed to label lysosomes of live cells in blue fluorescence. The kit uses a proprietary lysotropic dye that selective-ly accumulates in lysosomes probably via the lysosome pH gradi-ent. The stain has Ex/Em = 350/440 nm. The lysotropic indicator is a hydrophobic compound that easily permeates intact live cells, and trapped in lysosomes after it gets into the cells. Its fluorescence is significantly enhanced upon entering lysosomes. This key feature significantly reduces its staining background. The labeling protocol is robust, requiring minimal hands-on time. The kit can be readily adapted for many different types of fluorescence platforms such as microplate assays, flow cytometry and fluorescence microscope. It is useful for a variety of studies, including cell adhesion, chemo-taxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. The kit provides all the essential components with an optimized cell-labeling protocol and can be used for both proliferating and non-proliferating cells (either suspension or adherent cells).

Cell Navigator™ Lysosomal Staining Kit *Green Fluorescence with 405 nm Excitation*


22651 1 kit < - 15 oC

Lysosomes are cellular organelles which contain acid hydrolase enzymes to break up waste materials and cellular debris. Lysosomes digest excess or worn-out organelles, food particles, and engulfed viruses or bacteria. The membrane around a lysosome allows the digestive enzymes to work at pH 4.5. The interior of the lysosomes is acidic (pH 4.5-4.8) compared to the slightly alkaline cytosol (pH 7.2). The lysosome maintains this pH differential by pumping protons (H+ ions) from the cytosol across the membrane via proton pumps and chloride ion channels.

This particular kit is designed to label lysosomes of live cells in green fluorescence of large Stokes Shift at Ex/Em = 405/520 nm. The kit uses a proprietary lysotropic dye that selectively accumulates in lysosomes probably via the lysosome pH gradient. The lysotropic indicator, a hydrophobic compound, easily permeates intact live cells and gets trapped inside the lysosomes. Its fluorescence is significantly enhanced upon entering lysosomes. This key feature significantly reduces its staining background and make it useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. The kit provides all the essential components. It is suitable for both suspension and adherent cells.

Figure 6.24. Image of U2OS cells stained with the Cell Navigator™ Lysosomal Staining Kit (22655) in a 96-well Costar black wall/clear bottom plate.

Related Products



22656 1 kit Cell Navigator™ Lysosomal Staining Kit *Green Fluorescence*

22658 1 kit Cell Navigator™ Lysosomal Staining Kit *Red Fluorescence*

22666 1 kit Cell Navigator™ Mitochondrial Staining Kit *Green Fluorescence*

22667 1 kit Cell Navigator™ Mitochondrial Staining Kit *Orange Fluorescence*

22668 1 kit Cell Navigator™ Mitochondrial Staining Kit *Red Fluorescence*

21204 1 mg RatioWorks™ PDMPO


168737j1_cat.indd 69 2/4/11 11:54 AM



Related Products


70


6

Lyso

som

al S

tain

ing

Cell Navigator™ Lysosomal Staining Kit *Green Fluorescence*


22656 1 kit < - 15 oC

Lysosomes are cellular organelles which contain acid hydrolase enzymes to break up waste materials and cellular debris. Lysosomes digest excess or worn-out organelles, food particles, and engulfed viruses or bacteria. The membrane around a lysosome allows the digestive enzymes to work at pH 4.5. The interior of the lysosomes is acidic (pH 4.5-4.8) compared to the slightly alkaline cytosol (pH 7.2). The lysosome maintains this pH differential by pumping protons from the cytosol across the membrane via proton pumps and chlo-ride ion channels.

This particular kit is designed to label lysosomes of live cells in green fluorescence at Ex/Em = 490/525 nm. The proprietary lysotropic dye used in the kit selectively accumulates in lysosomes probably via the lysosome pH gradient. The lysotropic indicator is a hydropho-bic compound that easily permeates intact live cells, and trapped in lysosomes after it gets into cells. Its fluorescence is significantly enhanced upon entering lysosomes. This key feature significantly reduces its staining background and make it useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. It is suitable for proliferat-ing and non-proliferating cells, and can be used for both suspension and adherent cells.

Cell Navigator™ Lysosomal Staining Kit *Red Fluorescence*


22658 1 kit < - 15 oC


This particular kit is designed to label lysosomes of live cells in red fluorescence. The kit uses a proprietary lysotropic dye that selective-ly accumulates in lysosomes probably via the lysosome pH gradi-ent. The red lysosomal stain used in the kit has Ex/Em = 550/590 nm. The lysotropic indicator is a hydrophobic compound that easily permeates intact live cells, and trapped inside lysosomes after it gets into cells. Its fluorescence is strongly enhanced upon entering lysosomes. This key feature significantly reduces its staining back-ground. The labeling protocol is robust, requiring minimal hands-on time. The kit can be readily adapted for many types of fluorescence platforms such as microplate assays, flow cytometry and fluores-cence microscope. It is useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. The kit provides all the essential components and it can be used for both proliferating and non-proliferating cells (either suspension or adherent cells).



Related Products



22655 1 kit Cell Navigator™ Lysosomal Staining Kit *Blue Fluorescence*

22651 1 kit Cell Navigator™ Lysosomal Staining Kit * Green Fluorescence with 405 nm Excitation*

22666 1 kit Cell Navigator™ Mitochondrial Staining Kit *Green Fluorescence*

22667 1 kit Cell Navigator™ Mitochondrial Staining Kit *Orange Fluorescence*

22668 1 kit Cell Navigator™ Mitochondrial Staining Kit *Red Fluorescence*

21204 1 mg RatioWorks™ PDMPO

168737j1_cat.indd 70 2/4/11 11:54 AM




6

Mitochondrial Staining

Cell Navigator™ Mitochondrial Staining Kit *Green Fluorescence*


22666 1 kit < - 15 oC

Mitochondria are membrane-enclosed organelles found in most eukaryotic cells. Mitochondria are sometimes described as “cellular power plants” because they generate most of the cellular supply of ATP. In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Mitochondria have been implicated in several human diseases, including mitochondrial disorders and cardiac dysfunc-tion, and may play a role in the aging process. Although most of a cellular DNA is contained in the cell nucleus, the mitochondrion has its own independent genome.

This particular kit is designed to label mitochondria of live cells in green fluorescence at Ex/Em = 490/520 nm. The kit uses a propri-etary dye that selectively accumulates in mitochondria probably via the mitochondrial membrane potential gradient. The mitochon-drial indicator, a hydrophobic compound, easily permeates intact live cells and trapped in mitochondria after it gets into cells. The mitochondrial indicator is retained in mitochondria for a long time since it carries a cell-retaining group. This key feature significantly increases the staining efficiency.

Cell Navigator™ Mitochondrial Staining Kit *Orange Fluorescence with 405 nm Excitation*


22673 1 kit < - 15 oC


This particular kit is designed to label mitochondria of live cells in orange fluorescence of large Stokes Shift at Ex/Em = 405/550 nm. The kit uses a proprietary dye that selectively accumulates in mitochondria probably via the mitochondrial membrane potential gradient. The mitochondrial indicator, a hydrophobic compound, easily permeates intact live cells and trapped in mitochondria after it gets into cells. This fluorescent mitochondrial indicator is retained in mitochondria for a long time since it carries a cell-retaining group. This key feature significantly increases the staining efficiency. The mitochondrial stain used in the kit is quite photostable, making the fluorescence quite robust. The kit provides all the essential com-ponents with an optimized cell-labeling protocol. It is suitable for proliferating and non-proliferating cells, and can be used for both suspension and adherent cells.

Figure 6.28. Image of U2OS cells stained with the Cell Navigator™ Mitochondrial Staining Kit (22666) in a 96-well Costar black wall/clear bottom plate.

Related Products


22801 1 kit Cell Meter™ JC-10 Mitochondrial Membrane Potential Assay Kit *Optimized for Flow Cytometry Assays*

22800 1 kit Cell Meter™ JC-10 Mitochondrial Membrane Potential Assay Kit *Optimized for Microplate Assays*

22802 1 kit Cell Meter™ NIR Mitochondria Membrane Potential Assay Kit *Optimized for Flow Cytometry*

22803 1 kit Cell Meter™ NIR Mitochondria Membrane Potential Assay Kit *Optimized for Microplate Reader*

22200 5 mg JC-1 [5,5,6,6-Tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide]

22204 5x100 µL JC-10 *Superior alternative to JC-1*

22210 25 mg Rhodamine 123

Figure 6.29. Image of Hela cells stained with the Cell Navigator™ Mitochondria Staining Kit (22673) in a 96-well Costar black wall/clear bottom plate.

168737j1_cat.indd 71 2/4/11 11:54 AM




6

Mito

chon

dria

l Sta

inin

g

Cell Navigator™ Mitochondrial Staining Kit *Orange Fluorescence*


22667 1 kit < - 15 oC

Mitochondria are membrane-enclosed organelles found in most eukaryotic cells. Mitochondria are sometimes described as “cellular power plants” because they generate most of the cellular supply of ATP. In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Mitochondria have been implicated in several human diseases, including mitochondrial disorders and cardiac dysfunc-tion, and may play a role in the aging process. Although most of a cellular DNA is contained in the cell nucleus, the mitochondrion has its own independent genome.

This particular kit uses a proprietary dye that selectively accumu-lates in mitochondria probably via the mitochondrial membrane potential gradient. The orange fluorescent mitochondrial stain has Ex/Em = 545/575 nm (TRITC filter-compatible). The mitochon-drial indicator, a hydrophobic compound, easily permeates intact live cells and trapped in mitochondria after it gets into cells. This fluorescent mitochondrial indicator is retained in mitochondria for a long time since it carries a cell-retaining group. This key feature significantly increases the staining efficiency. The kit can be readily adapted for many different types of fluorescence platforms, such as microplate assays, immunocytochemistry and flow cytometry.

Cell Navigator™ Mitochondrial Staining Kit *Red Fluorescence*


22668 1 kit < - 15 oC


This particular kit is designed to label mitochondria in live cells with red fluorescence. The kit uses our proprietary dye that selectively accumulates in mitochondria probably via the mitochondrial membrane potential gradient. The red fluorescent mitochondrial stain used in the kit has Ex/Em = 580/600 nm (Texas Red® filter-com-patible) (Texas Red® is the trademark of InVitrogen). The mitochon-drial indicator, a hydrophobic compound, easily permeates intact live cells and trapped in mitochondria after it gets into cells. This fluorescent mitochondrial indicator is retained in mitochondria for a long time since it carries a cell-retaining group. This key feature significantly increases the staining efficiency. The kit can be readily adapted for many different types of fluorescence platforms, such as microplate assays, immunocytochemistry and flow cytometry. It is useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. The kit provides all the essential components and can be used for both proliferating and non-proliferating cells.

Figure 6.30. Image of U2OS cells stained with the Cell Navigator™ Mitochondrial Staining Kit (22667) in a 96-well Costar black wall/clear bottom plate.

Figure 6.31. Image of U2OS cells stained with the Cell Navigator™ Mitochondrial Staining Kit (22668) in a 96-well Costar black wall/clear bottom plate

Related Products









168737j1_cat.indd 72 2/4/11 11:54 AM


Cellular Function Analysis

Cellular Function Analysis7

168737j1_cat.indd 73 2/4/11 11:54 AM



www.aatbio.com Cellular Function Analysis

Cellu

lar F

unct

ion

Ana

lysi

s

7

cellular function assay kits at-a-glance*

Cell Functions Flow Cytometry Imaging Microplate

Apoptosis Assay (Caspase 3/7 Activity) 22823 22823 22795 & 22796

Apoptosis Assay (Generic Caspase Activity) 22821 & 22822 22821 & 22822

Apoptosis Assay (Caspase 8 Activity) 22798

Apoptosis Assay (Caspase 9 Activity) 22799

Apoptosis Assay (Intracellular GSH) 22810

Apoptosis Assay (Nuclear) 22811 22811

Apoptosis Assay (Phosphatidylserine) 22831, 22832, 22835 & 22836 22791, 22792, 22793 & 22794 22791, 22792, 22793 & 22794

Calcium Assay (Fluo-8) 36308, 36309, 36315 & 36316 36308, 36309, 36315 & 36316

Calcium Assay (Luminometric) 36305 & 36306

Calcium Assay (Ratiometric) 36320 & 36321

Calcium Assay (Rhod-4) 36331, 36332, 36334 & 36335 36331, 36332, 36334 & 36335

cAMP Assay (Colorimetric ELISA) 36370

cAMP Assay (Fluorimetric ELISA) 36373

cAMP Assay (Fluorescence Polarization) 36376 & 36377

cAMP Assay (FRET) 36379 & 36380

cAMP Assay (Live Cell) 36382

Cell Viability Assay 22785, 22786 & 22789 22785, 22786 & 22789

Cell Cycle Assay (Fluorimetric) 22841 & 22842

Cell Cytotoxicity Assay (Colorimetric) 22780

Cell Cytotoxicity Assay (Fluorimetric) 22781

Chloride Channel Assay 36350

Hydrogen Peroxide Assay 11503 11503

MDR Assay 36341 36341

Membrane Potential Assay 36005 & 36006

Mitochondrial Memberane Potential Assay 22801, 22802 & 22804 22800, 22803 & 22805 22800, 22803 & 22805

ROS Activity Assay 22900 & 22901 22900 & 22901 22900 & 22901




Cat # Size Storage ConditionCat # Size Storage Condition Cat # Size Storage ConditionCat # Size Storage Condition Cat # Size Storage ConditionCat # Size Storage Condition Cat # Size Storage ConditionCat # Size Storage Condition

75



7

Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit * Blue Fluorescence*


22795 1 kit < - 15 oC

Our Cell Meter™ assay kits are a set of tools for monitoring cellular functions. A variety of parameters can be used to monitor cell func-tions. This particular kit is designed to monitor cell apoptosis by measuring Caspase 3/7 activation. Caspase 3/7 is widely accepted as a reliable indicator for cell apoptosis since the activation of cas-pase 3 (CPP32/apopain) is important for the initiation of apoptosis. Caspase 3/7 is proven to have substrate selectivity for the peptide sequence Asp-Glu-Val-Asp (DEVD).

This kit uses Ac-DEVD-AMC as a fluorogenic indicator for caspase 3 activity. Cleavage of AMC peptides by Caspase 3/7 generates strongly fluorescent AMC that is monitored at the emission between 440 nm and 460 nm with the excitation at 350 nm. It is robust and can be readily adapted for high throughput screenings. The kit can be used to either quantify the activated caspase 3/7 activities in apoptotic cells or screen the caspase 3/7 inhibitors. The kit provides an optimized assay protocol and comes with the essential compo-nents as follows:

• AMC caspase substrate• Assay buffer

Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit * Green Fluorescence*


22796 1 kit < - 15 oC

There are a variety of parameters that can be used for monitoring cell apoptosis. Caspase 3/7 is widely accepted as a reliable indica-tor for cell apoptosis since the activation of caspase 3/7 (CPP32/apopain) is important for the initiation of apoptosis. Caspase 3/7 is proven to have substrate selectivity for the peptide sequence Asp-Glu-Val-Asp (DEVD). This particular kit is designed to track cell apoptosis by measuring caspase 3/7 activation.

This Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit uses Z-DEVD-R110-DVED-Z as a fluorogenic indicator for detecting caspase 3/7 activity. Cleavage of Rh110 peptides by caspase 3/7 generates strongly fluorescent Rh110 which is monitored at the emission between 520 nm and 530 nm with the excitation between 480 nm and 500 nm. The caspase 3/7-generated product has strong fluo-rescence with good photostability. The kit provides all the essential components with an optimized assay protocol. The assay is robust and can be readily adapted for high throughput screenings. It can be used to either quantify the activated caspase 3/7 activities in apoptotic cells or screen the caspase 3/7 inhibitors.

Figure 7.1. Detection of caspase 3/7 activities in Jurkat cells. Jurkat cells were seeded on the same day at 80,000 cells/well/90 µL in a 96-well black wall/clear bottom Costar plate. The cells were treated with or without 20 µM of camptothecin for 5 hours, and/or 5 µM of the caspase inhibitor AC-DEVD-CHO for 10 minutes. The caspase 3/7 assay solution (100 µL/well) was added and incubated at room tempera-ture for 1 hour. The fluorescence intensity was measured at Ex/Em = 350/450 nm

Figure 7.2. Detection of caspase 3/7 activities in Jurkat cells. Jurkat cells were seeded on the same day at 80,000 cells /well/90 µL in a 96-well black wall/clear bottom costar plate. The cells were treated with or without 20 µM of camptothecin for 5 hours, and/or 5 µM of the caspase inhibitor AC-DEVD-CHO for 10 minutes. The caspase 3/7 assay solution (100 µL/well) was added and incubated at room tempera-ture for 1 hour. The fluorescence intensity was measured at Ex/Em = 490/525 nm.

Related Products


22795 1 kit Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit * Blue Fluorescence*


13407 1 mg FITC-C6-LEHD-FMK

13431 1 mg (Ac-IETD)2-R110

13430 1 mg (Z-DEVD)2-R110

13420 5 mg Z-DEVD-AFC

13421 5 mg Z-DEVD-AMC

Caspase 3/7

168737j1_cat.indd 75 2/4/11 11:55 AM




Related Products


76


Cellular Function Analysis www.aatbio.comCa

spas

e/Ca

spas

e 3/

7

Cell Meter™ Caspase 3/7 Activity Assay Kit *Green Fluorescence Optimized for Flow Cytometry*


22823 1 kit < - 15 oC

The activation of caspase 3 (CPP32/apopain) is important for the initiation of apoptosis. It has been proven that caspase 3/7 has sub-strate selectivity for the peptide sequence Asp-Glu-Val-Asp (DEVD). This kit uses TF2-DEVD-FMK as a fluorescent indicator for caspase 3/7 activity. TF2-DEVD-FMK, which is cell permeable and nontoxic, irreversibly binds to activated caspase 3/7 in apoptotic cells. Once bound to caspase 3/7, the fluorescent reagent is retained inside the cell. The binding event prevents the caspase 3/7 from further cataly-sis but will not stop apoptosis from proceeding. Within 15 minutes after being added into the medium, the reagent will start to react with active caspase 3/7 enzymes.

There are a variety of parameters that can be used for monitoring cell apoptosis. This Cell Meter™ Caspase 3/7 Activity Assay Kit is de-signed to detect cell apoptosis by measuring caspase 3/7 activation in live cells. It is used for the quantification of activated caspase 3/7 activities in apoptotic cells, or for screening caspase 3/7 inhibitors. TF2-DEVD-FMK, the green label reagent, allows for direct detection of activated caspase 3/7 in apoptotic cells by flow cytometry. The kit provides all the essential components with an optimized assay protocol.

Cell Meter™ Generic Caspase Activity Assay Kit *Green Fluorescence Optimized for Flow Cytometry*


22821 1 kit < - 15 oC

Our Cell Meter™ assay kits are a set of tools for monitoring cellular functions. The activation of caspase is widely accepted as a reliable indicator for cell apoptosis. Most caspases have substrate selectivity for the peptide sequence Val-Ala-Asp (VAD). This kit uses TF2-VAD-FMK as a fluorescent indicator for most caspase activities. The cell permeable and nontoxic TF2-VAD-FMK irreversibly binds to acti-vated casepase-1, -3, -4, -5, -6, -7, -8 and -9 in apoptotic cells. Once bound to caspases, the fluorescent reagent is retained inside the cell. The binding event prevents the caspases from further catalysis but will not stop apoptosis from proceeding. Within 15 minutes incubation, it starts to react with active caspase enzymes.

This Cell Meter™ Generic Caspase Activity Assay Kit provides all the essential components with an optimized assay protocol. It is designed to detect cell apoptosis by measuring generic activation of caspases (caspase-1, -3, -4, -5, -6, -7, -8 and -9) in live cells. The kit is used for either the quantification of most activated caspase activities in apoptotic cells or screening of caspase inhibitors. TF2-VAD-FMK, the green label reagent, allows for direct detection of activated caspases in apoptotic cells by flow cytometry at Ex/Em = 488/520 nm.

Figure 7.3. Detection of caspase 3/7 activities using the Kit 22823 in Jurkat cells. TF2-DEVD-FMK fluorescence intensity is induced by the addition of Camptothecin. Jurkat cells were treated without (Blue) or with 20 μM camptothecin (Red) in a 37 oC, 5% CO2 incubator for 4-5 hours, and then loaded with TF2-DEVD-FMK for 1 hour.

Figure 7. 4. The detection of caspase activities using Kit 22821 in Jurkat cells. TF2-VAD-FMK fluores-cence intensity is induced with the addition of camptothecin in Jurkat cells. Jurkat cells were treated without (Blue) or with 20 μM camptothecin (Red) in a 37 oC, 5% CO2 incubator for 4-5 hours, and then loaded with TF2-VAD-FMK for 1 hour.

Related Products





13431 1 mg (Ac-IETD)2-R110

13430 1 mg (Z-DEVD)2-R110



7

168737j1_cat.indd 76 2/4/11 11:55 AM



Related Products


77


7

Caspase/Caspase 8

7

Cell Meter™ Generic Caspase Activity Assay Kit *Red Fluorescence Optimized for Flow Cytometry*


22822 1 kit < - 15 oC

This particular kit is designed to monitor cell apoptosis through measuring generic caspases (caspase-1, -3, -4, -5, -6, -7, -8 and -9) activation in live cells with our red fluorescent TF5-VAD-FMK probe. Most caspases have substrate selectivity for the peptide sequence Val-Ala-Asp (VAD). The cell permeable and nontoxic TF5-VAD-FMK irreversibly binds to activated casepase-1, -3, -4, -5, -6, -7, -8 and -9 in apoptotic cells. Once bound to caspases, the fluorescent reagent is retained inside the cell. The binding event prevents the caspases from further activation but will not stop apoptosis from proceeding.

With the spectral properties almost identical to those of Cy5® or Alexa Fluor® 647 (Ex/Em = ~647/660 nm), TF5-VAD-FMK can be conveniently used with the common fluorescence instruments equipped with the light sources and filters for Cy5® or Alexa Fluor® 647 (Cy5® or Alexa Fluor® 647 are the trademarks of GE Healthcare and Invitrogen respectively). The red label allows for direct detec-tion of activated caspases in apoptotic cells by flow cytometry. It can be used for the quantification of most activated caspases activi-ties in apoptotic cells, or for the screening of caspases inhibitors. The kit provides all the essential components.

Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Green Fluorescence*


22798 1 kit < - 15 oC

Our Cell Meter™ assay kits are a set of tools for monitoring cellular functions. There are a variety of parameters that can be used to monitor cell apoptosis. This particular kit is designed to monitor cell apoptosis by measuring caspase 8 activity. Caspase 8 is a caspase protein, encoded by the CASP8 gene. Caspase 8 also plays an important role in neurodegenerative diseases such as Huntington disease.

Caspase 8 is proven to have substrate selectivity for the peptide sequence Ile-Glu-Thr-Asp (IETD). This kit uses (Ac-IETD)2-R110 as a fluorogenic indicator for caspase 8 activity. Cleavage of rhodamine 110 (R110) peptides by caspase 8 generates strongly fluorescent R110 which is monitored at the emission between 520 nm and 530 nm with the excitation between 480 nm and 500 nm. This spectral feature makes the kit compatible with the FITC filter set. The kit provides all the essential components with an optimized assay protocol. The assay can be readily adapted for high throughput screenings. It can be used to either quantify the activated caspase 8 activities in apoptotic cells or screen the caspase 8 inhibitors.

Figure 7.5. Detection of caspase activities using Kit 22822 in Jurkat cells. TF5-VAD-FMK fluorescence intensity is induced with the addition of camptothecin. Jurkat cells were treated without (Blue) or with 20 μM camptothecin (Red) in a 37 oC, 5% CO2 incubator for 4-5 hours, and then incubated with TF5-VAD-FMK for 1 hour.

Related Products





13431 1 mg (Ac-IETD)2-R110

13430 1 mg (Z-DEVD)2-R110



Figure 7.6. Detection of caspase 8 activities using Kit 22798 in Jurkat cells. Jurkat cells were seeded on the same day at 200,000 cells/90 μL/well in a 96-well black wall/clear bottom Costar plate. The cells were treated with 20 μM camptothecin for 4 hours while the untreated cells were used as control. The caspase 8 assay solution (100 μL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured at Ex/Em = 490/525 nm with a FlexStation™ microplate reader.

168737j1_cat.indd 77 2/4/11 11:55 AM



Related Products


78


spas

e 9/

GSH

Cell Meter™ Intracellular GSH Assay Kit *Optimized for Flow Cytometry*


22810 1 kit < - 15 oC

There are a variety of parameters that can be used for monitoring cell apoptosis. This particular kit is designed to detect cell apoptosis by measuring the decrease in reduced glutathione (GSH). GSH is important for maintaining redox level of cells. It is involved in many cellular processes including the scavenging of free radicals, drug detoxification, cell signaling, and cell proliferation. The decrease in cellular GSH concentration is an early hallmark in the progression of cell death in response to different apoptotic stimuli in many cells.

Our Cell Meter™ Intracellular GSH Assay Kit uses our proprietary non-fluorescent Thiolite™ Green, which becomes strongly fluores-cent upon reacting with thiol (including GSH in cells). In normal cells, Thiolite™ Green is accumulated primarily in cytosol, but it is partially translocated to mitochondria in apoptotic cells while Thiolite™ Green staining intensity is decreased. Cells stained with Thiolite™ Green can be visualized with flow cytometry at Ex/Em = 490/520 nm (FL1 channel). The kit can be used together with other reagents, such as 7-AAD (#17501) for multi-parametric study of cell viability and apoptosis. The kit is optimized for screening apoptosis activators and inhibitors with flow cytometry.

Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Green Fluorescence*


22799 1 kit < - 15 oC

Our Cell Meter™ assay kits are a set of tools for monitoring cellular functions. There are a variety of parameters that can be used. This particular kit is designed to monitor cell apoptosis by measuring caspase 9 activity. Caspase 9 is a member of the CED-3 subfam-ily. Activated Caspase-9 cleaves downstream caspases such as caspase-3, -6 and -7, initiating the caspase cascade. It is essential for apoptosis during normal development of the central nervous system.

Caspase 9 is proven to have selectivity for the peptide sequence Leu-Glu-His-Asp (LEHD). This kit uses (Ac-LEHD)2-R110 as a fluoro-genic indicator for caspase 9 activity. Cleavage of R110 peptides by caspase 9 generates strongly fluorescent rhodamine 110 (R110)which is monitored at the emission between 520 nm and 530 nm with the excitation between 480 nm and 500 nm. The kit provides all the essential components. The assay is robust and can be readily adapted for high throughput screening. It can be used to either quantify the activated caspase 9 activities in apoptotic cells or screen the caspase 9 inhibitors. Quite a few labs have used this kit for high throughput screenings.

Figure 7.8. The decrease in the fluorescence intensity of Thiolite™ Green adduct with the addition of camptothecin in Jurkat cells. Jurkat cells were treated overnight without (blue) or with 20 μM camptothecin (red) in a 37 oC, 5% CO2 incubator, and loaded with Thiolite™ Green for 30 minutes. The fluorescence intensity of Thiolite™ Green was measured with a FACSCalibur flow cytometer using FL1 channel.

Related Products









Figure 7.7. Detection of caspase 9 activities using kit 22799 in Jurkat cells. Jurkat cells were seeded at 200,000/cells/90 μL/well in a 96-well black wall/clear bottom Costar plate. The cells were treated with 10 mM adenosine for 48 hours while the untreated cells were used as control. The caspase 9 assay solution (100 μL/well) was added and incubated at room temperature for 1hour. The fluorescence intensity was measured at Ex/Em = 490/525 nm with a FlexStation™ microplate reader.

7

168737j1_cat.indd 78 2/4/11 11:55 AM




Related Products


79



7

Nuclear A

poptosis/Phosphatidylserine

7

Cell Meter™ Nuclear Apoptosis Assay Kit*Green Fluorescence*


22811 1 kit < - 15 oC

This particular kit is designed to monitor cell apoptosis by mea-suring the apoptotic chromatin condensation. The compacted chromatin of apoptotic cells binds higher amounts of nuclear dye compared to the healthy cells. The fluorometric assay is based on the detection of the DNA contents in cells using our proprietary non-fluorescent dye that becomes strongly fluorescent upon bind-ing to cellular DNA.

In normal cells, Nuclear Green™ DCS1 is not cell permeable. How-ever, in apoptotic cells, Nuclear Green™ DCS1 can easily get into the cells with compromised plasma membranes. Once inside the cell, the dye binds to intracellular DNA producing highly fluorescent complexes which identify the cells as non-viable cells. The staining density with Nuclear Green™ DCS1 can be measured with a flow cytometer at Ex/Em = 490/520 nm (FL1 channel) or a fluorescence microscope (FITC filter set). The kit can be used together with our other apoptosis reagents, such as our Cell Meter™ NIR Mitochondrial Membrane Potential Detection Kit (#22802), for multi-parametric study of cell viability and apoptosis. The kit is optimized for screen-ing apoptosis activators and inhibitors.

Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Green Fluorescence Optimized for Microplate Readers*


22791 1 kit < - 15 oC

Our Cell Meter™ assay kits are a set of tools for monitoring cellular functions. There are a variety of parameters that can be used for monitoring cell viability. This particular kit is designed to monitor cell apoptosis by measuring the translocation of phosphatidylserine (PS). In apoptosis, PS is transferred to the outer leaflet of the plasma membrane. The appearance of phosphatidylserine on the cell sur-face is a universal indicator of the initial/intermediate stages of cell apoptosis and can be detected before morphological changes can be observed.

Compared to Annexin V, the Apopxin™ PS sensor used in this kit is reengineered to have a much higher affinity to PS with Kd < 10 nM. The PS sensor has green fluorescence upon binding to membrane PS. The fluorescent sensor has Ex/Em = 490/520 nm, making assay readily run with FITC filter set. Due to its highly enhanced affinity to PS, this kit is more robust than the other commercial Annexin V-based apoptosis kits that are only used with either microscope or flow cytometry platform. This kit can be used with a fluorescence microplate reader besides the microscope and flow cytometry plat-forms. It has been used for HTS applications.

Figure 7.10. Detection of phosphatidylserinen binding activity in Jurkat cells. Jurkat cells were seeded on the same day at 200,000 cells/90µL/well in a 96-well black wall/clear bottom Costar plate. The cells were treated with different doses of camptothecin for 5 hours as indicated. The Apopxin™ Green assay solution (100 µL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured at Ex/Em = 490/525 nm with NOVOstar instrument (BMG Labtech).

Related Products



22796 1 kit Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit * Green Fluorescence*






Figure 7.9. Increase in fluorescence intensity of Nuclear Green™ DCS1 with the addition of Camptoth-ecin in Jurkat cells. Jurkat cells were treated overnight without (A) or with 20 μM camptothecin (B) in a 37 oC, 5% CO2 incubator, and then dye loaded with Nuclear Green™ DCS1 for 60 minutes. At the end of 15 minutes of Nuclear Green™ DCS1 dye loading, MitoLite™ NIR (22802) was added for multicolor analysis. The fluorescence intensity of Nuclear Green™ DCS1 and MitoLite™ NIR was measured with a FACSCalibur flow cytometer using FL1 channel and FL4 channel.

A B

168737j1_cat.indd 79 2/4/11 11:55 AM




Related Products


80


Cellular Function Analysis www.aatbio.comPh

osph

atid

ylse

rine

Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Red Fluorescence*


22792 1 kit < - 15 oC

Our Cell Meter™ assay kits are a set of tools for monitoring cellular functions. There are a variety of parameters that can be used for monitoring cell viability. This particular kit is designed to monitor cell apoptosis by measuring the translocation of phosphatidylserine (PS). In apoptosis, PS is transferred to the outer leaflet of the plasma membrane. The appearance of phosphatidylserine on the cell sur-face is a universal indicator of the initial/intermediate stages of cell apoptosis and can be detected before morphological changes can be observed.

Compared to Annexin V, the Apopxin™ PS sensor used in this kit is reengineered to have a much higher affinity to PS with Kd < 10 nM. The PS sensor has red fluorescence upon binding to membrane PS. The red fluorescent sensor has Ex/Em = ~590/630 nm with good photostability. The spectral property is compatible with Texas Red® filter set. Due to its highly enhanced affinity to PS, this kit is more ro-bust than the other commercial Annexin V-based apoptosis kits that are only used with either microscope or flow cytometer platform. This kit can be used with a fluorescence microplate reader besides the microscope and flow cytometry platforms. It has been used for HTS applications.

Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Orange Fluorescence*


22794 1 kit < - 15 oC

This particular kit is designed to monitor cell apoptosis through measuring the translocation of phosphatidylserine (PS). In apopto-sis, PS is transferred to the outer leaflet of the plasma membrane. The appearance of phosphatidylserine on the cell surface is a universal indicator of the initial/intermediate stages of cell apop-tosis and can be detected before morphological changes can be observed.

This kit uses our proprietary orange fluorescent Apopxin™ PS sensor that specifically binds PS with affinity much higher than Annexin V (Kd < 10 nM). The PS sensor used in this kit has orange fluorescence upon binding to membrane PS. The stain has the spectral proper-ties almost identical to those of Cy3® or Alexa Fluor® 555, making it convenient to be used for the common fluorescence instruments equipped with the light sources and filters for Cy3® or Alexa Fluor® 555 (Cy3® or Alexa Fluor® 555 are the trademarks of GE Healthcare and Invitrogen respectively). Due to its highly enhanced affinity to PS, this kit is more robust than the other commercial Annexin V-based apoptosis kits that are only used with either microscope or flow cytometry platform. This kit can be also used with a fluores-cence microplate reader besides the microscope and flow cytom-etry platforms.

Related Products







22802 1 kit Cell Meter™ NIR Mitochondrial Membrane Potential Assay Kit *Optimized for Flow Cytometry*

22803 1 kit Cell Meter™ NIR Mitochondrial Membrane Potential Assay Kit *Optimized for Microplate Reader*

A B

Figure 7.12. Images of Jurkat cells in a 96-well Costar black wall/clear bottom plate stained with the Cell Meter™ Phosphatidylserine Apoptosis Assay Kit (22792). A: Untreated control cells B: Cells treated with 20 µM camptothecin for 5 hours

7

A B

Figure 7.11. Images of Jurkat cells in a 96-well Costar black wall/ clear bottom plate stained with the Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Orange Fluorescence* A: Untreated control cells B: Cells treated with 20 µM camptothecin for 5 hours

168737j1_cat.indd 80 2/4/11 11:55 AM




Related Products


81



7

Phosphatidylserine

7

Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Deep Red Fluorescence*


22793 1 kit < - 15 oC


This kit uses our proprietary red fluorescent Apopxin™ PS sensor that specifically binds PS with affinity much higher than Annexin V (Kd < 10 nM). The PS sensor used in this kit has red fluorescence upon binding to membrane PS. The stain has the spectral proper-ties almost identical to those of Cy5® or Alexa Fluor® 647, making it convenient to be used with the common fluorescence instruments equipped with the light sources and filters for Cy5® or Alexa Fluor® 647 (Cy5® or Alexa Fluor® 647 are the trademarks of GE Healthcare and Invitrogen respectively). Due to its highly enhanced affinity to PS, this kit is more robust than the other commercial Annexin V based apoptosis kits that are only used with either microscope or flow cytometry platform. This kit can be also used with a fluores-cence microplate reader besides the microscope and flow cytom-etry platforms.

Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Blue Fluorescence with 405 nm Excitation*


22835 1 kit < - 15 oC

Our Cell Meter™ assay kits are a set of tools for monitoring cellular functions. There are a variety of parameters that can be used for monitoring cell viability. This particular kit is designed to monitor cell apoptosis through measuring the translocation of phosphati-dylserine (PS). In apoptosis, PS is transferred to the outer leaflet of the plasma membrane. The appearance of phosphatidylserine on the cell surface is a universal indicator of the initial/intermediate stages of cell apoptosis and can be detected before morphological changes can be observed.

Compared to Annexin V, the Apopxin™ PS sensor used in this kit is reengineered to have a much higher affinity to PS with Kd < 10 nM. The PS sensor has blue fluorescence upon binding to membrane PS. The PS stain used in the kit has the spectral properties similar to those of Pacific Blue® at Ex/Em = ~400/450 nm (Pacific Blue® is the trademark of Invitrogen). The blue fluorescent stain is well excited with the Violet Laser at 405 nm, and emits intense blue fluorescence at ~450 nm. The kit is optimized to be used with a flow cytometer equipped with a Violet Laser. It is particularly suitable for multicolor flow cytometric analysis of cells. This kit has been used for flow cy-tometric analysis of cells in multicolor applications in combination with fluorescent antibodies.

A B

Figure 7.13. Images of Jurkat cells in a 96-well Costar black wall/clear bottom plate stained with the Cell Meter™ Phosphatidylserine Apoptosis Assay Kit (22793). A: Untreated control cells B: Cells treated with 20 µM of camptothecin for 5 hours.

Related Products







22802 1 kit Cell Meter™ NIR Mitochondrial Membrane Potential Assay Kit *Optimized for Flow Cytometry*

22803 1 kit Cell Meter™ NIR Mitochondrial Membrane Potential Assay Kit *Optimized for Microplate Reader*

Figure 7.14. Detection of phosphatidylserine binding activity in Jurkat cells. Jurkat cells were treated without (Blue) or with 20 μM camptothecin (Red) in a 37°C, 5% CO2 incubator for 4-5 hours, and then dye loaded with Apopxin™ Violet 450 for 1hour. The fluorescence intensity of Apopxin™ Violet 450 was measured with a FACSCalibur flow cytometer with Violet laser.

168737j1_cat.indd 81 2/4/11 11:55 AM




82


Cellular Function Analysis www.aatbio.com

Related Products








22835 1 kit Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Blue Fluorescence Excited at 405 nm*

Pho

spha

tidyl

serin

e

Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Green Fluorecence with 405 nm Excitation*

Cat. # Size Storage Condition

22836 1 kit < - 15 oC

This particular kit is designed to monitor cell apoptosis by measur-ing the translocation of phosphatidylserine (PS). In apoptosis, PS is transferred to the outer leaflet of the plasma membrane. The appearance of phosphatidylserine on the cell surface is a universal indicator of the initial/intermediate stages of cell apoptosis and can be detected before morphological changes can be observed.

This kit uses our proprietary green fluorescent Apopxin™ PS sensor that specifically binds PS with affinity much higher than Annexin V (Kd < 10 nM). The PS sensor used in this kit has green fluorescence upon binding to membrane PS. The green fluorescent stain is well excited with the Violet Laser at 405 nm, and emits intense green fluorescence at ~520 nm. The kit is optimized to be used with a flow cytometer equipped with Violet Laser. It is particularly suitable for multicolor flow cytometric analysis of cells. In coupling with its large Stokes Shift, its highly enhanced affinity to PS makes this kit more robust than the other commercial Annexin V based apoptosis kits that are only used with either microscope or flow cytometry plat-form. This kit can be also used with a fluorescence microplate reader besides the microscope and flow cytometry platforms.

Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Green Fluorescence Optimized for Flow Cytometry*


22831 1 kit < - 15 oC

This particular kit is designed to monitor cell apoptosis by measur-ing the translocation of phosphatidylserine (PS). In apoptosis, PS is transferred to the outer leaflet of the plasma membrane. The appearance of phosphatidylserine on the cell surface is a universal indicator of the initial/intermediate stages of cell apoptosis and can be detected before morphological changes can be observed.

This kit uses our proprietary green fluorescent Apopxin™ PS sensor that specifically binds PS with affinity much higher than Annexin V (Kd < 10 nM). The PS sensor used in this kit has green fluorescence upon binding to membrane PS. The stain has the spectral properties almost identical to those of FITC with much higher photostability, making it convenient to be used with the common fluorescence instruments equipped with the light sources and filters for FITC, the most common fluorophore. This kit is optimized for flow cytometric applications while Kit 22791 is optimized to be used with a fluores-cence microplate reader or a fluorescence microscope. This kit is a convenient alternative to Annexin V-FITC conjugate. The kit provides all the essential components with an optimized protocol for flow cytometric applications.

Figure 7.16. Detection of phosphatidylserine binding activity in Jurkat cells. Jurkat cells were treated without (Blue) or with 20 μM camptothecin (Red) in a 37 oC, 5% CO2 incubator for 4-5 hours, and then loaded with Apopxin™ Green for 15 minutes. The fluorescence intensity of Apopxin™ Green was mea-sured with a FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer in FL1 channel.

7

Figure 7.15. Detection of phosphatidylserine binding activity in Jurkat cells. Jurkat cells were treated without (Blue) or with 20 μM camptothecin (Red) in a 37°C, 5% CO2 incubator for 4-5 hours, and then dye loaded with Apopxin™ Violet 500 for 1hour. The fluorescence intensity of Apopxin™ Violet 500 was measured with a FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer with Violet laser.

168737j1_cat.indd 82 2/4/11 11:55 AM




83



7Related Products





22835 1 kit Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Blue Fluorescence Excited at 405 nm*

22793 1 kit Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Deep Red Fluorescence*


21120 1 mg Quest Rhod-4™, AM

Phosphatidylserine/Calcium

7

Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Red Fluorescence Optimized for Flow Cytometry*


22832 1 kit < - 15 oC


This kit uses our proprietary red fluorescent Apopxin™ PS sensor that specifically binds PS with affinity much higher than Annexin V (Kd < 10 nM). The PS sensor used in this kit has red fluorescence upon binding to membrane PS. The stain has the spectral proper-ties almost identical to those of Cy5® or Alexa Fluor® 647, making it convenient to be used with the common fluorescence instruments equipped with the light sources and filters for Cy5® or Alexa Fluor® 647 (Cy5® or Alexa Fluor® 647 are the trademarks of GE Healthcare and Invitrogen respectively). This kit is optimized for flow cytometric applications while Kit 22792 is optimized to be used with a fluores-cence microplate reader or a fluorescence microscope.

Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kits


36307 1 plate < - 15 oC

36308 10 plates < - 15 oC

36309 100 plates < - 15 oC

Screen Quest™ Fluo-8 NW Calcium Assay Kits provide homoge-neous fluorescence-based assays for detecting intracellular calcium mobilization. Cells expressing a GPCR of interest that signals through calcium are pre-loaded with Fluo-8 NW which can cross cell membrane. Once inside the cell, the lipophilic blocking groups of Fluo-8 NW are cleaved by esterase, resulting in a negatively charged fluorescent dye that stays inside cells. Its fluorescence is greatly enhanced upon binding to calcium. When cells stimulated with agonists, the receptor signals the release of intracellular calcium, which significantly increase the fluorescence of Fluo-8 NW. The char-acteristics of its long wavelength, high sensitivity, and >100 times fluorescence enhancement make Fluo-8 NW an ideal indicator for the measurement of cellular calcium. The Screen Quest™ Fluo-8 NW Calcium Assay Kits can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation.

Figure 7.18. Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Fluo-8 NW Assay kit and Fluo-4 NW Assay Kit. HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. The growth medium was removed, and the cells were incubated with 100 µL of dye-loading solution using the Screen Quest™ Fluo 8-NW calcium assay kit or the Fluo-4 NW kit (according to the manufacturer’s instructions) for 1 hour at room temperature. Carbachol (25µL/well) was added by NOVOstar to achieve the final indicated concentrations. The fluorescence signal was measured at Ex/Em = 490/525 nm. The EC50 of Fluo-8 NW is about 1.2 µM.

Figure 7.17. Detection of phosphatidylserine binding activity in Jurkat cells. Jurkat cells were treated without (Blue) or with 20 μM camptothecin (Red) in a 37°C, 5% CO2 incubator for 4-5 hours, and then dye loaded with Apopxin™ Red for 1hour. The fluorescence intensity of Apopxin™ Red was measured with a FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer in FL4 Channel.

168737j1_cat.indd 83 2/4/11 11:55 AM



Related Products


84


lciu

m

Screen Quest™ Fluo-8 No Wash Calcium Assay KitsCat # Size Storage Condition

36314 1 plate < - 15 oC

36315 10 plates < - 15 oC

36316 100 plates < - 15 oC

Screen Quest™ Fluo-8 NW Calcium Assay Kits provide homogeneous fluorescence-based assays for detecting intracellular calcium mobili-zation. Cells expressing a GPCR of interest that signals through calci-um are pre-loaded with Fluo-8 NW which can cross cell membrane. Once inside the cell, the lipophilic blocking groups of Fluo-8 NW are cleaved by esterases, resulting in a negatively charged fluores-cent dye that stays inside cells. Its fluorescence is greatly enhanced upon binding to calcium. When cells stimulated with agonists, the receptor signals the release of intracellular calcium, which signifi-cantly increase the fluorescence of Fluo-8 NW. The characteristics of its long wavelength, high sensitivity, and >100 times fluorescence enhancement make Fluo-8 NW an ideal indicator for the measure-ment of cellular calcium. The Screen Quest™ Fluo-8 NW Calcium Assay Kits provide an optimized assay method for monitoring the G-protein-coupled receptors and calcium channels. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation.

Screen Quest™ Luminometric Calcium Assay KitsCat # Size Storage Condition

36305 10 plates < - 15 oC

36306 100 plates < - 15 oC

Calcium flux assays are preferred methods in drug discovery for screening G protein coupled receptors (GPCR). These kits use a highly calcium-sensitive and membrane-permeable coelenterazine analog as a calcium indicator for the cells that are transfected with apoaequorin gene. Aequorin is a calcium-sensitive bioluminescent protein from the jellyfish Aequorea victoria that has been used extensively as a calcium indicator in cells. The aequorin complex emits blue light when bound to calcium ions. The luminescence intensity is directly proportional to the Ca2+ concentration. Our coelenterazine-based kits are much more sensitive than the fluores-cence-based calcium assay kits (such as Fluo-4, Fluo-3, Calcium-3 and Calcium-4). These kits provide an optimized assay method for monitoring the G-protein-coupled receptors and calcium channels. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation. It might be useful for monitoring the intracellular calcium mobilization in a specified compartment given that recombinant apoaequorin pro-teins can now be targeted to specific organelles, cells and tissues. This kit is more sensitive than the fluorescent calcium assays.

Figure 7.20. ATP Dose Response on CHO-aeq cells. CHO cells stably transfected with apoaequrin were seeded overnight at 50,000 cells/100 µL/well in a 96-well white wall/clear bottom Costar plate. The growth medium was removed and the cells were incubated with 100 µL of dye-loading solution using the Screen Quest™ Luminometric Calcium Assay Kit for 3 hours at room temperature with light protection. ATP (25 µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations. The EC50 of ATP is about 0.8 µM.

Figure 7.19. Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Fluo-8 No Wash Calcium Assay Kit and Fluo-4 NW Calcium Assay Kit. HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. The cells were incubated with 100 µL of dye-loading solution using the Screen Quest™ Fluo-8 No Wash calcium assay kit or Fluo-4 NW kit (according to the manufacturer’s instructions) for 1 hour at room temperature. Carbachol (50µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations. The fluorescence signal was measured at Ex/Em = 490/525 nm.

Related Products


21150 250 µg Coelenterazine *UltraPure grade*

21153 250 µg Coelenterazine h *UltraPure grade*

21154 250 µg Coelenterazine hcp *UltraPure grade*


21090 1 mg Quest Fluo-8H™, AM

21097 10x50 µg Quest Fluo-8L™, AM


7

168737j1_cat.indd 84 2/4/11 11:55 AM




7

Calcium

7

Screen Quest™ Ratiometric Fura-2 No Wash Calcium Assay Kit Cat # Size Storage Condition

36320 10 plates < - 15 oC

36321 100 plates < - 15 oC

Calcium flux assays are preferred methods in drug discovery for screening G protein coupled receptors (GPCR). Screen Quest™ Ra-tiometric Calcium Assay Kit provides a homogeneous fluorescence-based assay for detecting the intracellular calcium mobilization. Cells expressing a GPCR of interest that signals through calcium are pre-loaded with Fura-2 AM which can cross cell membrane. Once in-side the cell, the lipophilic blocking groups of Fura-2 Am are cleaved by non-specific cell esterases, resulting in a negatively charged fluorescent dye which stays inside the cells. The 340/380 nm excita-tion ratio allows accurate measurements of the intracellular Ca2+

concentration. The characteristics of its ratio measurements reduce the effects of uneven dye loading and cell numbers, dye leakage and photo bleaching. This Screen Quest™ Ratiometric Calcium Assay Kit provides an optimized assay method with no washing step for monitoring the G-protein-coupled receptors and calcium channels. The assay can be performed in a convenient 96-well or 384-well microtiter plate format and easily adapted to automation. The kit comes with all the essential components with an optimized protocol to use with FlexStation or an equivalent instrument.

Related Products


21010 1 mg Fluo-3, AM

21020 1 mg Fura-2, AM

21030 1 mg Indo-1, AM





Screen Quest™ Rhod-4 No Wash Calcium Assay KitsCat # Size Storage Condition

36333 1 plate < - 15 oC

36334 10 plates < - 15 oC

36335 100 plates < - 15 oC

Screen Quest™ Rhod-4 NW Calcium Assay Kits provide a homo-geneous fluorescence-based assay for detecting the intracellular calcium mobilization. Cells expressing a GPCR of interest that signals through calcium are pre-loaded with our proprietary Rhod-4 NW which can cross cell membrane. Rhod-4 is the brightest red calcium indicator available for HTS screening. Once inside the cell, the lipophilic blocking groups of Quest Rhod-4™ are cleaved by non-specific cell esterases, resulting in a negatively charged fluorescent dye that stays inside the cells. Its fluorescence is greatly enhanced upon binding to calcium. When cells stimulated with screening compounds, the receptor signals the release of intracellu-lar calcium, which greatly increase the fluorescence of Rhod-4. This Screen Quest™ Rhod-4 NW Calcium Assay Kit provides an optimized assay method for monitoring the G-protein-coupled receptors and calcium channels. The kit comes with all the essential components with an optimized protocol to use with FLIPR® or FDSS™ or an equivalent instrument.

Figure 7.22. Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Rhod-4 NW Assay kit and Rhod-2 AM. HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a 96-well black wall/clear bottom Costar plate. The cells were incubated with 100 µL of dye-loading solution using the Screen Quest™ Rhod-4 NW calcium assay kit, or 100 µL of Rhod-2 AM solution (5 μM) for 1 hour at room temperature. Carbachol (25µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations. The fluorescence signal was measured at Ex/Em = 540/590 nm. The EC50 of Rhod-4 NW is about 0.6 μM.

Figure 7.21. ATP Dose Response in CHO cells measured with Screen Quest™ Ratiometric Calcium As-say Kit. CHO-K1 cells were seeded overnight at 40,000 cells per 100 µL per well in a 96-well black wall/clear bottom Costar plate. The cells were incubated with 100 µL of the Screen Quest™ Ratiometric Cal-cium Assay Kit for 1 hour at room temperature. ATP (50µL/well) was added by a FlexStation (Molecular Devices) to achieve the final indicated concentrations.

168737j1_cat.indd 85 2/4/11 11:55 AM




7

Calc

ium

/cA

MP

Screen Quest™ Rhod-4 No Wash Calcium Assay Kits *Medium Removal*


36330 1 plate < - 15 oC

36331 10 plates < - 15 oC

36332 100 plates < - 15 oC

Cells expressing a GPCR of interest that signals through calcium are pre-loaded with our proprietary Rhod-4 NW which can cross cell membrane. Quest Rhod-4™ is the brightest red calcium indica-tor available for HTS screening. Once inside the cell, the lipophilic blocking groups of Quest Rhod-4™ are cleaved by non-specific cell esterases, resulting in a negatively charged fluorescent dye which stays inside the cells. Its fluorescence is greatly enhanced upon binding to calcium. When cells stimulated with screening com-pounds, the receptor signals release of intracellular calcium, which significantly increase the fluorescence of Quest Rhod-4™. The char-acteristics of its long wavelength, high sensitivity, and >250 times fluorescence increases make Quest Rhod-4™ an ideal indicator for the measurement of cellular calcium. These Screen Quest™ Rhod-4 NW Calcium Assay Kits provide an optimized assay method for monitoring the G-protein-coupled receptors and calcium channels. Compared to Fluo-3, Fluo-4 and Quest Fluo-8™, Quest Rhod-4™ is more photostable, making its fluorescence imaging more robust.

Figure 7.23. Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Rhod-4 NW Assay kit and Rhod-2 AM. HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. The growth medium was removed, and the cells were incubated with 100 µL of the dye-loading solution using the Screen Quest™ Rhod-4 NW calcium assay kit, or 100 µL of Rhod-2 AM solution (5 μM) for 1 hour at room temperature. Carbachol (25µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations.

Related Products


21010 1 mg Fluo-3, AM

21020 1 mg Fura-2, AM

21030 1 mg Indo-1, AM





7

Screen Quest™ Colorimetric ELISA cAMP Assay Kit


36370 1 plate < - 15 oC

36371 10 plates < - 15 oC

Adenosine 3’, 5’ cyclic monophosphate (cAMP) is an important second messenger in intracellular signal transduction. Monitoring levels of cAMP is one of the most common ways to screen for ago-nists and antagonists of GPCRs. Screen Quest™ cAMP Colorimetric ELISA Assay Kit is based on the competition between HRP-labeled cAMP and non-labeled cAMP. HRP-cAMP is displaced from the HRP-cAMP/anti-cAMP antibody complex by unlabeled free cAMP. In the absence of cAMP, HRP-cAMP conjugate is bound to anti-cAMP antibody exclusively. However, the unlabeled free cAMP in the test sample competes for anti-cAMP antibody with the HRP-cAMP antibody conjugate, therefore inhibits the binding of HRP-cAMP to anti-cAMP antibody.

Our Screen Quest™ cAMP Colorimetric ELISA Assay Kit provides a sensitive method for detecting adenylate cyclase activity in biochemical or cell-based assay system. Compared to other ELISA cAMP assay kits, our kit eliminates the tedious acetylation step. The kit uses Amplite™ Blue as a colorimetric substrate to quantify the HRP activity. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format.

Figure 7.24. cAMP dose response was measured with Screen Quest™ cAMP Colorimetric ELISA Assay Kit in a 96-well clear plate with a SpectraMax microplate reader (Molecular devices). As low as 0.1 nM cAMP was detected in a 100 µL reaction.

168737j1_cat.indd 86 2/4/11 11:55 AM




Related Products


36370 1 kit Screen Quest™ Colorimetric ELISA cAMP Assay Kit

36308 1 kit Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit *Optimized for Difficult Cell Lines*

36315 1 kit Screen Quest™ Fluo-8 No Wash Calcium Assay Kit

36373 1 kit Screen Quest™ Fluorimetric ELISA cAMP Assay Kit

36382 1 kit Screen Quest™ Live Cell cAMP Assay Serive Pack

36334 1 kit Screen Quest™ Rhod-4 No Wash Calcium Assay Kit *1% FBS Growth Medium*

36331 1 kit Screen Quest™ Rhod-4 No Wash Calcium Assay Kit *Medium Removal*

7

cAM

P

Screen Quest™ Fluorimetric ELISA cAMP Assay Kit


36373 1 plate < - 15 oC

36374 10 plates < - 15 oC

Screen Quest™ cAMP Fluorescence ELISA Assay Kit provides an optimized assay method for monitoring the activation of adenylyl cyclase in G-protein coupled receptor systems. The assay is based on the competition for a fixed number of antibody binding sites between HRP-labeled cAMP and non-labeled cAMP. HRP-cAMP is displaced from the HRP-cAMP/anti-cAMP antibody complex by unlabeled free cAMP. In the absence of cAMP, HRP-cAMP conjugate is bound to anti-cAMP antibody exclusively. However, the unlabeled free cAMP in the test sample competes for anti-cAMP antibody with the HRP-cAMP antibody conjugate, therefore inhibits the binding of HRP-cAMP to anti-cAMP antibody.

Our Screen Quest™ cAMP Fluorometric Assay Kit provides a sensitive method for detecting adenylate cyclase activity. Compared to other commercial ELISA cAMP assay kits, this cAMP assay kit only requires a single wash step to remove unbound material prior to the devel-opment step. It also eliminates the tedious acetylation step. The kit uses Amplite™ Red as a fluorogenic HRP substrate to quantify the HRP activity. The fluorescent product formed is proportional to the activity of HRP-cAMP conjugate.

Figure 7.25. cAMP dose response was measured with Screen Quest™ cAMP Fluorescence ELISA Assay Kit in a 96-well solid black plate with a Gemini microplate reader (Molecular Devices). As low as 0.1 nM cAMP was detected in a 100 µL reaction.

Screen Quest™ Fluorimetric No Wash cAMP Assay Kits*Fluorescence Polarization*


36376 1 plate < - 15 oC

36377 10 plates < - 15 oC

Adenosine 3’, 5’ cyclic monophosphate (cAMP) is an important sec-ond messenger in intracellular signal transduction. Screen Quest™ No-Wash cAMP Fluorescence Polarization Assay Kit provides the most convenient assay method for monitoring the activation of adenylyl cyclase in G-protein coupled receptor systems. Compared to other commercial ELISA cAMP assay kits, this homogenous cAMP assay kit does not require a wash step or the acetylation step. The assay is based on the competition for a fixed number of anti-cAMP antibody binding sites between the fluorescent cAMP tracer and non-labeled free cAMP. Free cAMP displaces the fluorescent cAMP tracer from the fluorescent cAMP/anti-cAMP antibody complex. In the absence of cAMP, fluorescent cAMP conjugate is bound to anti-cAMP antibody exclusively. The free cAMP in cAMP test sample competes for anti-cAMP antibody with the fluorescent cAMP tracer, therefore inhibits the binding of the fluorescent cAMP tracer to anti-cAMP antibody. The unbound fluorescent cAMP tracer rotates faster with low MP (polarization value) while anti-cAMP-bound fluores-cent cAMP tracer has much higher MP value. The magnitude of MP change is proportional to the concentration of cAMP in a sample.

Figure 7.26. cAMP dose response was measured with Screen Quest™ cAMP Fluorescence Polarization Assay Kit in a 96-well solid black plate with a NOVOstar plate reader (BMG Labtech). As low as 5 nM cAMP was detected in a 100 µL reaction.

168737j1_cat.indd 87 2/4/11 11:55 AM




Screen Quest™ FRET No Wash cAMP Assay Kit Cat # Size Storage Condition

36379 1 plate < - 15 oC

36380 10 plates < - 15 oC

As our kit 36376, Screen Quest™ No Wash FRET cAMP Assay Kit provides another convenient assay method for monitoring the ac-tivation of adenylyl cyclase in G-protein coupled receptor systems. Compared to other commercial ELISA cAMP assay kits, this homog-enous cAMP assay kit does not require a wash step or the acetyla-tion step. The assay is based on the competition for a fixed number of anti-cAMP antibody binding sites between the fluorescent cAMP tracer and non-labeled free cAMP. Free cAMP displaces the fluores-cent cAMP tracer from the HRP-cAMP/anti-cAMP antibody complex. The anti-cAMP antibody is labeled with our TR Fluor™ Tb while the cAMP tracer contains our bFluor™ 650. In the absence of cAMP, bFluor™ 650-cAMP conjugate is bound to TR Fluor™ Tb-labeled anti-cAMP antibody exclusively to have a strong FRET. However, the unlabeled free cAMP in the test sample competes for the TR Fluor™ Tb-labeled anti-cAMP antibody conjugate, therefore inhibits the binding of bFluor™ 650-cAMP to anti-cAMP antibody. The bFluor™ 650 labeled cAMP tracer only has fluorescence lifetime of nanosec-ond while TR Fluor™ Tb-labeled anti-cAMP antibody-bound fluo-rescent cAMP tracer has much longer fluorescence lifetime value due to the TR-FRET. The magnitude of FRET is proportional to the concentration of cAMP in a sample. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format.

Figure 7.27. cAMP dose response was measured with Screen Quest™ FRET No Wash cAMP Assay Kit in a 96-well solid black plate. As low as 0.3 nM cAMP was detected in a 100 µL reaction.

cAM

P/Ce

ll Vi

abili

ty

Screen Quest™ Live Cell cAMP Assay Service PackCat # Size Storage Condition

36382 1 kit < - 60 oC

G protein coupled receptors (GPCR) are one of the largest receptor classes targeted by drug discovery programs. Calcium flux (coupled via Gq pathway) assay is a preferred method in drug discovery for screening GPCR targets. However, over 60% of the known GPCRs signal through adenylyl cyclase activity coupled to cAMP. Most of the existing cAMP assays not only require cell lysis but also lack both temporal and spatial resolution. Screen Quest™ Live Cell cAMP Assay Service Pack provides the real-time monitoring of intracel-lular cAMP change in a high-throughput format without a cell lysis step. The assay works through the cell lines that contain either an exogenous cyclic nucleotide-gated channel(CNGC) or the promiscu-ous G-protein, Gα16. The channel is activated by elevated levels of intracellular cAMP, resulting in ion flux and cell membrane depo-larization which can be detected with either a fluorescent calcium (such as Quest Fluo-8 AM™) or a fluorescent membrane potential dye. Co-expression of Gα16with specific non-Gq-coupled receptors will result in the generation of an intracellular calcium signal upon receptor stimulation. The Screen Quest™ Live Cell cAMP Assay Service Pack provides both cell lines and reagents for the measure-ment of intracellular cAMP changes with a FLIPR, a FDSS or other equivalent fluorescence microplate readers. It has been successfully used to measure Gs and Gi coupled GPCR activity.

Related Products


38000 2 x106 cells Screen Quest™ Amylin 3 Receptor

38001 2 x106 cells Screen Quest™ Cannabinoid Receptor

38002 2 x106 cells Screen Quest™ Chemokine (C-C) Receptor 2B

38003 2 x106 cells Screen Quest™ Chemokine (C-X-C motif ) Receptor 4

38004 2 x106 cells Screen Quest™ Dopamine Receptor 1 (DRD1)

38005 2 x106 cells Screen Quest™ Glucagon-like Receptor 1 (GLP1R)

38006 2 x106 cells Screen Quest™ Opiate Receptor-like 1 (ORL1)

7Figure 7.28. Screen Quest™ Live Cell cAMP Assay Principle



www.aatbio.com Cellular Function AnalysisCell Viability

7

Cell Meter™ Cell Viability Assay Kit *Blue Fluorescence*


22785 1 kit < - 15 oC

The Cell Meter™ assay kits are a set of tools for monitoring cell vi-ability and cellular functions. There are a variety of parameters that can be used to monitor cell viability. This kit uses our proprietary cell viability dye whose fluorescence is strongly enhanced upon enter-ing into live cells. The dye is a hydrophobic compound that easily permeates intact live cells. The weakly fluorescent CytoCalcein™ Violet 450, AM is hydrolyzed by intracellular esterase to generate a strongly fluorescent hydrophilic product that is well-retained in the cell cytoplasm. The esterase activity is proportional to the number of viable cells, and thus directly related to the fluorescence intensity of the product generated from the esterase-catalyzed hydrolysis of the fluorogenic substrate. Cells grown in black wall/clear bottom plates can be stained and quantified in less than two hours. The assay is more robust than tetrazolium salt or Alarmar Blue® based ones. It can be readily adapted for many different types of fluorescence platforms such as microplate assays, fluorescence microscope, and flow cytometry. The kit is useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. It provides all the essential components with an optimized cell-labeling protocol and can be used for both suspension and adherent cells.

Figure 7.29. CHO-K1 cell number response was measured with Cell Meter™ Cell Viability Kit. CHO-K1 cells at 0 to 5,000 cells/well/100 µL were seeded overnight in a 96-well black wall/clear bottom Costar plate. The cells were incubated with 100 µL/well of CytoCalcein™ Violet 450, AM dye-loading solution for 1 hour at room temperature. The fluorescence intensity was measured at Ex/Em = 405/460 nm.

Cell Meter™ Cell Viability Assay Kit *Green Fluorescence*


22786 1 kit < - 15 oC

There are a variety of parameters that can be used to monitor cell viability. The proprietary green fluorescent dye used in the kit is a hydrophobic compound. It easily permeates intact live cells and gets enhanced fluorescence upon entering into live cells. The hydrolysis of the non-fluorescent substrate by intracellular esterases generates a strongly green fluorescent hydrophilic product that is well-retained in the cell cytoplasm. The green fluorophore generat-ed by the non-fluorescent substrate used in the kit has the spectral properties of fluorescein at Ex/Em = ~490 nm/520 nm. When well excited with the Argon Laser at 488 nm, the fluoreophore emits intense green fluorescence at ~520 nm.

The kit provides all the essential components with an optimized cell-labeling protocol for fluorescence microplate assays. It can also be used with a fluorescence microscope equipped with a FITC filter set. This Cell Meter™ Cell Viability Assay Kit provides an effective tool of labeling cells for fluorescence microplate and microscopic investigations of cellular functions. It is useful for a variety of stud-ies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. The kit is suitable for proliferat-ing and non-proliferating cells.

Figure 7.30. CHO-K1 cell number response was measured with Cell Meter™ Cell Viability Assay Kit. CHO-K1 cells at 0 to 5,000 cells/well/100 µL were seeded overnight in a 96-well black wall/clear bottom Costar plate. The cells were incubated with 100 µL/well of CytoCalcein™ Green dye-loading solution for 1 hour at 37 oC. The fluorescence intensity was measured at Ex/Em = 490/ 525 nm.

Related Products



22007 1 mg Calcein blue, AM

22006 25 mg Calcein blue *UltraPure Grade*





168737j1_cat.indd 89 2/4/11 11:55 AM




Cell Meter™ Cell Viability Assay Kit *Green/Red Dual Fluorescence*


22789 1 kit < - 15 oC

The Cell Meter™ assay kits are a set of tools for monitoring cell viability and cellular functions. This particular kit uses two non-fluorescent indicators: CytoCalcein™ Green for viable cells and a cell-impermeable DNA-binding dye for the cells with compromised membranes. The non-fluorescent CytoCalcein™ Green can easily permeate intact live cells and is hydrolyzed by intracellular ester-ase to generate the strongly fluorescent hydrophilic CytoCalcein™ Green which is well-retained in the cell cytoplasm. The esterase activity is proportional to the number of viable cells. The DNA-bind-ing dye is quite polar and impermeable for viable cells that have intact membranes. It becomes fluorescent only upon binding to the DNA of dead cells. Cells grown in black-wall plates can be stained and quantified in less than two hours. The assay is more robust and accurate than the other viability assays. It can be readily adapted for a wide variety of fluorescence platforms such as microplate assays, fluorescence microscope, and flow cytometry. The kit provides all the essential components with an optimized assay protocol. It is suitable for both proliferating and non-proliferating cells (either suspension or adherent cells). This Cell Meter™ Cell Viability Assay kit comes with sufficient reagents to perform either 200 assays (96-well format) or 800 assays (a 384-well format).

Figure 7.31. The Effect of Saponin in Jurkat cells measured with Cell Meter™ Cell Viability Assay Kit. Jurkat cells at 2X106 cells/mL were treated with or without 0.5% Saponin for 5 minutes. The cells were centrifuged and the supernatant were replaced with fresh medium. 100 uL of untreated cells (A), 50 uL of untreated and 50 uL treated cells (B), 25 uL of untreated and 75 uL treated cells (C), and 100 uL of 0.5% saponin treated cells (D) were plated in a 96-well black wall/clear bottom Poly-D-lysine plate. The cells were incubated with 100 μL/well of CytoCalcein™ Green/ Propidium Iodide dye-loading solution for 1 hr at 37oC. The ratio of 490/525 nm to 540/650 nm fluorescence intensity on live and dead cells were showed as indicated (n=6).

Cell Meter™ Cell Viability Assay Kit *NIR Fluorescence Opti-mized for Fluorescence Microplate Reader*


22787 1 kit < - 15 oC

The proprietary far red fluorescent dye used in the kit is a hydropho-bic compound that can easily permeate intact live cells and gets en-hanced fluorescence upon entering into live cells. The hydrolysis of the weakly fluorescent substrate by intracellular esterases generates a strongly red fluorescent hydrophilic product that is well-retained in the cell cytoplasm. The red fluorophore generated by the weakly fluorescent substrate has the spectral properties similar to those of Cy5® or Alexa Fluor® 647 at Ex/Em = ~640/660 nm (Cy5® or Alexa Fluor® 647 are the trademarks of GE Healthcare and Invitrogen re-spectively). When well excited with the He-Ne Laser at 633 nm, the red fluorophore emits intense red fluorescence at ~660 nm.

This Cell Meter™ Cell Viability Assay Kit is an effective tool of label-ing cells for the investigations of cellular functions. It is useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. The kit is suit-able for proliferating and non-proliferating cells, and can be used for both suspension and adherent cells. It comes with reagents sufficient to perform 200 assays in a 96-well plate or 800 assays in a 384-well plate. The kit provides all the essential components with an optimized protocol for microplate reader analysis of cell viability.

Figure 7.32. CHO-K1 cell number response was measured with Cell Meter™ Cell Viability Assay Kit. CHO-K1 cells at 0 to 50,000 cells/well/100 µL were seeded overnight in a 96-well black wall/clear bot-tom Costar plate. The cells were incubated with 100 µL/well of CytoCalcein™ NIR dye-loading solution for 1 hour at room temperature. The fluorescence intensity was measured at Ex/Em = 635/670 nm with FlexStation™ microplate reader (Molecular Devices).

Related Products


17501 1 mg 7-AAD [7-Aminoactinomycin D]

17507 2 mL DAPI [4,6-Diamidino-2-phenylindole, dihydrochloride] *10 mM solution in water*

17530 100 mg Hoechst 33342 *UltraPure grade*

17550 0.5 mL Nuclear Green™ DCS1

17551 0.5 mL Nuclear Orange™ DCS1

17540 0.5 mL Nuclear Green™ LCS1

17541 0.5 mL Nuclear Orange™ LCS1

7

Cell

Viab

ility

168737j1_cat.indd 90 2/4/11 11:55 AM




7

Cell Cycle

Cell Meter™ Fluorimetric Cell Cycle Assay Kit *Green Fluorescence Optimized for Flow Cytometry*


22841 1 kit < - 15 oC

The cell cycle has four sequential phases: G0/G1, S, G2, and M. During a cell’s passage through cell cycle, its DNA is duplicated in S (synthesis) phase and distributed equally between two daughter cells in M (mitosis) phase. These two phases are separated by two gap phases: G0/G1 and G2. The two gap phases provide time for the cell to grow and double the mass of their proteins and organelles. They are also used by the cells to monitor internal and external conditions before proceeding with the next phase of cell cycle. The cell’s passage through cell cycle is controlled by a host of different regulatory proteins.

This particular kit is designed to monitor cell cycle progression and proliferation by using our proprietary Nuclear Green™ LCS1 in per-meabilized and fixed cells. The percentage of cells in a given sample that are in G0/G1, S and G2/M phases, as well as the cells in the sub-G1 phase prior to apoptosis can be determined by flow cytometry. Cells stained with Nuclear Green™ LCS1 can be monitored with a flow cytometer at Ex/Em = 490 nm/520 nm (FL1 channel).

Figure 7.33. DNA profile in growing and camptothecin treated Jurkat cells. Jurkat cells were treated without (red) or with 20 μM camptothecin (blue) in a 37 oC, 5% CO2 incubator for about 8 hours, and then loaded with Nuclear Green™ LCS1 for 60 minutes. The fluorescence intensity of Nuclear Green™ LCS1 was measured with a FACSCalibur flow cytometer in FL1 channel. In growing Jurkat cells, nuclear stained with Nuclear Green™ LCS1 shows G1, S, and G2 phases (red). In camptothecin treated apop-totic cells (Blue), both S and G2 phases were diminished.

Cell Meter™ Fluorimetric Cell Cycle Assay Kit *Red Fluorescence Optimized for Flow Cytometry*


22842 1 kit < - 15 oC

In normal cells, DNA density changes depending on whether the cell is growing, dividing, resting, or performing its ordinary func-tions. The progression of the cell cycle is controlled by a complex interplay among various cell cycle regulators. These regulators activate transcription factors, which bind to DNA and turn on or off the production of proteins resulted in cell division. Any misstep in this regulatory cascade causes abnormal cell proliferation which underlies many pathological conditions, such as tumor formation.

This particular kit is designed to monitor cell cycle progression and proliferation using Nuclear Red™ CCS1, a cell cycle stain in fixed cells. The dye passes through a permeabilized membrane and intercalates into cellular DNA. The signal intensity of Nuclear Red™ CCS1 is directly proportional to DNA content after RNA is degraded by RNase provided in the kit. The percentage of cells in a given sample that are in G0/G1, S and G2/M phases, as well as the cells in the sub-G1 phase prior to apoptosis can be monitored with a flow cytometer at Ex/Em = 490/620 nm (FL2 channel).

Figure 7.34. DNA profile in growing Jurkat cells. Jurkat cells were dye-loaded with Nuclear Red™ CCS1 and RNase A for 30 minutes. The fluorescence intensity of Nuclear Red™ CCS1 was measured with the FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer in FL2 channel.

Related Products


17501 1 mg 7-AAD [7-Aminoactinomycin D]

17513 25 mg DAPI [4,6-Diamidino-2-phenylindole, dihydrochloride]



17540 0.5 ml Nuclear Green™ LCS1

17541 0.5 ml Nuclear Orange™ LCS1

17515 25 mg Propidium iodide *UltraPure grade*

168737j1_cat.indd 91 2/4/11 11:55 AM



Cellular Function Analysis www.aatbio.comCe

ll Cy

toto

xici

ty

Cell Meter™ Colorimetric Cell Cytotoxicity Assay KitCat # Size Storage Condition

22780 1 kit < - 15 oC

Monitoring cell cytotoxicity is one of the most essential tasks for studying cellular functions. Our Cell Meter™ assay kits are a set of tools for monitoring cell viability. There are a variety of parameters that can be used. This kit uses a proprietary water-soluble dye that changes its absorption spectra upon cellular reduction. The absorp-tion ratio change is directly proportional to the number of living cells. The characteristics of its high sensitivity, non-radioactivity and no-wash method make the kit suitable for high throughput screening of cell proliferation or cytotoxicity against a variety of compounds.

This kit does not require pre-mixing of components and has higher sensitivity compared to the tetrazolium based colorimetric assays (such as MTT and XTT). It comes with reagents sufficient to run 1000 assays (regular size) or 5000 assays (bulk package). The kit com-ponents are quite stable with minimal cytotoxicity, thus a longer incubation time (such as 24 to 48 hours) is possible if required. Our Cell Meter™ Colorimetric Cell Cytoxicity Assay Kit is robust and convenient to use. It can be readily adapted for a wide variety of instrument platforms. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format.

Figure 7.35. CHO-K1 cell number response was measured with Cell Meter™ Colorimetric Cell Cyto-toxicity Assay Kit. CHO-K1 cells at 0 to 10,000 cells/well/100 µL were seeded overnight in a 96-well black wall/clear bottom Costar plate. The cells were incubated with 20 µL/well of Component A for 3 hours at 37 oC. The absorbance intensity was measured at 570 nm and 605 nm with SpectraMax plus (Molecular Devices). The ratio of OD570/OD605 is proportional to the number of cells as indicated.

Related Products



22007 1 mg Calcein blue, AM

22006 25 mg Calcein blue *UltraPure Grade*





Cell Meter™ Fluorimetric Cell Cytotoxicity Assay KitCat # Size Storage Condition

22781 1 kit < - 15 oC

The measurement of mitochondrial dehydrogenases (e.g. LDH) activity is a well-accepted assay to quantify cell numbers and moni-tor cell viability. Our Cell Meter™ Fluorimetric Cell Cytotoxicity Assay Kit provides a fast, simple, accurate and homogeneous assay for the colorimetric or fluorimetric detection of viable cells. This assay is based on the observation that oxidized non-fluorescent blue resazurin is reduced to a red fluorescent dye (resorufin) by accept-ing an electron from mitochondrial respiratory chain in live cells. The amount of resorufin produced is directly proportional to the number of living cells.

Our Cell Meter™ Fluorimetric Cell Cytotoxicity Assay Kit is more sen-sitive for cell proliferation and cytotoxicity than other assays such as MTT. The kit components are quite stable with minimal cytotoxicity, thus a longer incubation time (such as 24 to 48 hours) is possible if required. The characteristics of its high sensitivity (<100 CHO cells), non-radioactivity and no-wash method make the kit suitable for high throughput screening of cell proliferation or cytotoxicity against a variety of compounds. The assay can be performed in a convenient 96-well and 384-well microtiter-plate format with a filter set of Ex/Em = ~540/590 nm.

Figure 7.36. CHO-K1 cell number response was measured with Cell Meter™ Fluorimetric Cell Cytotox-icity Assay Kit. CHO-K1 cells at 0 to 10,000 cells/well/100 µL were seeded overnight in a 96-well black wall/clear bottom Costar plate. The cells were incubated with 20 µL/well of Component A for 3 hours at 37 oC. The fluorescence intensity was measured at Ex/Em = 540/590 nm with NOVOstar instrument (BMG Labtech). The fluorescence intensity was linear (R2 = 0.998) to the cell number as indicated. The detection limit is 60 cells/well (n=6).

7

168737j1_cat.indd 92 2/4/11 11:55 AM




7

Chloride Channel/Hydrogen Peroxide

Screen Quest™ Colorimetric Chloride Channel Assay Kit


36350 1 kit 2 - 6 oC

Chloride channels have a variety of important physiological and cellular functions that include regulation of pH, volume homeo-stasis, organic solute transport, cell migration, cell proliferation and differentiation. Chloride channels represent valuable drug targets. A number of chronic diseases such as cystic fibrosis and Bartter’s syndrome are due to defects in chloride channel functions. However, the existing technologies used to screen chloride channel modulators have to compromise between throughput, sensitivity and physiological relevance.

Screen Quest™ Colorimetric Chloride Channel Assay Kit provides a sensitive and robust colorimetric method for the study of chloride channels. The assay is based on our proprietary iodide indicator (Iodide Blue™) to measure iodide concentration, as low as 100 nM of iodide can be detected. Iodide Blue™ forms a blue complex with iodide, which has absorption spanning from the UV to 700 nm. Thus a few absorption wavelengths can be used for monitoring the iodide-dependent color change. Screen Quest™ Colorimetric Chlo-ride Channel Assay Kit provides an optimized assay for monitoring chloride channels. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format.

Figure 7.37. NaI dose response was measured with Screen Quest™ Colorimetric Chloride Channel Assay Kit in a 96-well black wall/clear bottom plate. As low as 100 nM of NaI was detected with 10 minutes incubation time (n=3).

Related Products



22900 1 kit Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Green Fluorescence*

22901 1 kit Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Orange Fluorescence*

21259 10 mg lucigenin [Bis-N-methylacridinium nitrate]

21250 100 mg MEQ [6-Methoxy-N-ethylquinolinium iodide]

21255 100 mg MQAE [N-(Ethoxycarbonylmethyl)-6-methoxyquinolinium bromide]

21252 25 mg SPQ [6-Methoxy-N-(3-sulfopropyl)quinolinium]

Amplite™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Green Fluorescence*


11503 1 kit < - 15 oC

Hydrogen peroxide (H2O2) is a reactive oxygen metabolic by-prod-uct that serves as a key regulator for a number of oxidative stress-related states. It is involved in many biological events that are linked to asthma, atherosclerosis, diabetic vasculopathy, osteoporosis, a number of neurodegenerative diseases and Down’s syndrome. The measurement of this reactive species is helpful for determining how oxidative stress modulates various intracellular pathways.

This Amplite™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit uses our unique ROS Green™ hydrogen peroxide sensor to quan-tify hydrogen peroxide in live cells. ROS Green™ is cell-permeable, and generates the green fluorescence when it reacts with hydrogen peroxide. The kit is an optimized “mix and read” assay format that is compatible with HTS liquid handling instruments. The Amplite™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit provides a sensitive, one-step fluorometric assay to detect as little as 0.3 nanomoles of H2O2 in a 100 µL assay volume (3 µM). The assay can be performed in a convenient 96-well or 384-well microtiter-plate format. Its signal can be easily read by either a fluorescence micro-plate reader at Ex/Em = 490/520 nm for H2O2 detection in solution or a fluorescence microscopy for live cell H2O2 imaging.

A B

Figure 7.38. Images of Live CHO-K1 cells in a 96-well Costar black wall/clear bottom plate. Live CHO-K1 cells were stained with Amplite™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit (11503). A: Control cells. B: Cells treated with 100 µM of H2O2 at room temperature for 5 minutes.

168737j1_cat.indd 93 2/4/11 11:55 AM



Related Products


94

Cellular Function Analysis www.aatbio.comM

DR

Ass

ay/M

embr

ane

Pote

ntia

l

Screen Quest™ Fluorimetric MDR Assay Kits


36340 1 plate < - 15 oC

36341 10 plates < - 15 oC

Multi-drug resistance (MDR) is a major factor in the failure of many forms of chemotherapy. In the past few years it has become widely accepted that the resistance to chemotherapy correlates with the overexpression of at least two ATP-dependent drug-efflux pumps. These cell membrane proteins, called P-glycoprotein (Pgp, MDR1), and multidrug-resistance-associated protein (MRP1) are members of the ABC transporter family. Our Screen Quest™ Fluorimetric MDR Assay Kits use a fluorescent MDR indicator for assaying these two MDR pump activities. This hydrophobic fluorescent dye molecule rapidly penetrates cell membranes and becomes trapped in cells. Following a short incubation, the intracellular free dye concentra-tion can increase significantly. In the MDR1 and/or MRP1-expressing cells this dye is extruded by the MDR transporters, thus decreasing the cellular fluorescence intensity. However, when its extrusion is blocked by an agent that interferes with the MDR1 and/or MRP1 pump-activity, its cellular fluorescence intensity increases signifi-cantly. The Screen Quest™ Fluorimetric MDR Assay Kits are ideal for high throughput screening of MDR pump inhibitors or identifying the cells that have high level of MDR pump activities.

Figure 7.39. Effect of Cyclosporin A on the inhibition of Pgp pump in MCF-7/ADR cells. The increased concentration of Cyclosporin A resulted in an increase in fluorescence signal caused by the inhibition of Pgp pump which enhanced the intracellular accumulation of MDR indicator dye. The EC50 = 2.4 μM (measured with the kit) is similar to the value reported in the literature.

Related Products





21414 25 mg DiSBAC2(3) [Bis-(1,3-diethylthiobarbituric acid)trimethine oxonol]

21483 1 mg MM 1-43 [N-(3-Triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide]

21489 1 mg MM 2-10 [N-(3-Triethylammoniumpropyl)-4-(4-(diethylamino)styryl)pyridinium dibromide]

21487 1 mg MM 4-64 [N-(3-Triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide]

Screen Quest™ Membrane Potential Assay Kit *Red Fluorescence*


36005 1 kit < - 15 oC

36006 1 kit < - 15 oC

Membrane potential is the difference in voltage between the interior and exterior of a cell. The membrane potential allows a cell to function as a battery, providing power to operate a variety of "molecular devices" embedded in the membrane. In electrically excitable cells such as neurons, membrane potential is used for transmitting signals between different parts of a cell. Opening or closing of ion channels at one point in the membrane produces a lo-cal change in the membrane potential, which causes electric current to flow rapidly to other points in the membrane. Ion channels have been identified as important drug discovery targets.

Our Screen Quest™ Membrane Potential Assay Kit is a homo-geneous assay with fast read time. It uses our proprietary long wavelength membrane potential indicator to detect the membrane potential change that is caused by the opening and closing of the ion channels. The red fluorescence of the membrane potential indicator used in the kit has enhanced fluorescence upon entering cells and minimizes the interferences resulted from the screening compounds and/or cellular autofluorescence.

Figure 7.40. ATP Dose Response in HEK cells transiently transfected with P2X receptor. HEK cells tran-siently transfected with P2X receptor were seeded overnight at 40,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. The cells were incubated with 100 µL of the MP dye-loading solu-tion in a 5% CO2, 37 oC incubator for 60 minutes. ATP (50 µL/well) was added by FlexStation to achieve the final indicated concentrations. The fluorescence signal was measured using the bottom read mode at Ex/Em = 620/650 nm (cutoff at 630 nm).

7

168737j1_cat.indd 94 2/4/11 11:56 AM



Related Products


95


7

Mitochondrial M

embrane Potential

7

Cell Meter™ JC-10 Mitochondrial Membrane Potential Assay Kit *Optimized for Flow Cytometry*


22801 1 kit < - 15 oC

Although JC-1 is widely used in many labs, its poor water solubility causes great inconvenience. JC-10 is developed to be a superior alternative to JC-1. Compared to JC-1, JC-10 has much better water solubility. JC-10 is capable of selectively entering into mitochondria, and reversibly changes its color from green to orange as membrane potentials increase. When excited at 490 nm, the color of JC-10 changes reversibly from green to orange as the mitochondrial membrane becomes more polarized. Both colors can be detected using the filters commonly mounted in all flow cytometers. The green emission can be analyzed in fluorescence channel 1 (FL1) and orange emission in channel 2 (FL2).

This kit is based on the detection of the mitochondrial membrane potential changes in cells by the cationic, lipophilic JC-10 dye. In normal cells, JC-10 concentrates in the mitochondrial matrix where it forms red fluorescent aggregates. However, in apoptotic and necrotic cells, JC-10 exists in monomeric form and stains cells green. The kit is optimized to screen both apoptosis activators and inhibi-tors using flow cytometry while Kit 22800 is offered for microplate-based high throughput screening applications.

Cell Meter™ JC-10 Mitochondrial Membrane Potential Assay Kit *Optimized for Microplate Reader Assays*


22800 1 kit < - 15 oC

Compared to JC-1, JC-10 has much better water solubility. JC-10 is capable of selectively entering into mitochondria, and reversibly changes its color from green to orange as membrane potentials increase. When excited at 490 nm, the color of JC-10 changes revers-ibly from green to orange as the mitochondrial membrane becomes more polarized. The green emission can be analyzed in fluorescence channel 1 (FL1) and orange emission in channel 2 (FL2).

This Cell Meter™ JC-10 Mitochondrial Membrane Potential Assay Kit enables researchers to run JC-10 assay in the format of microplate reader, and it provides the most robust assay method for monitor-ing mitochondria membrane potential changes. The kit is based on the detection of the mitochondrial membrane potential changes in cells by the cationic, lipophilic JC-10 dye. In normal cells, JC-10 con-centrates in the mitochondrial matrix where it forms red fluorescent aggregates. However, in apoptotic and necrotic cells, JC-10 diffuses out of mitochondria. It changes to monomeric form and stains cells in green fluorescence. The kit can be used to screen both apoptosis activators and inhibitors. The assay can be performed in a conve-nient 96-well and 384-well fluorescence microtiter-plate format.

Figure 7.41. Effect of FCCP induced mitochondria membrane potential change in JurKat cells. Jurkat cells were loaded with JC-10 dye-loading solution along with DMSO (Top) or 5 μM FCCP (Low) for 10 minutes. The fluorescent intensities for both J-aggregates and monomeric forms of JC-10 were mea-sured with a FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer using FL1 and FL2 channels. Uncompensated data (left column) were compared with compensated data (right column).

Figure 7.42. Campotothecin induced mitochondria membrane potential changes were measured with JC-10 and JC-1 in Jurkat cells. After Jurkat cells were treated with camptothecin (10 µM) for 4 hours, JC-1 and JC-10 dye loading solutions were added to the wells and incubated for 30 minutes. The fluorescent intensities for both J-aggregates and monomeric forms of JC-1 and JC-10 were mea-sured at Ex/Em = 490/525 nm and 490/590 nm with NOVOstar microplate reader (BMG Labtech).

Related Products


22804 1 kit Cell Meter™ Mitochondrial Membrane Potential Assay Kit*Orange Fluorescence Optimized for Flow Cytometry*




22211 10 mg Rhodamine B, hexyl ester, perchlorate

22220 25 mg TMRE [Tetramethylrhodamine ethyl ester]

22221 25 mg TMRM [Tetramethylrhodamine methyl ester]

Control

FCCP

Uncompensated Compensated

168737j1_cat.indd 95 2/4/11 11:56 AM




Related Products





22204 5 x 100 uL JC-10 *Superior alternative to JC-1*


22220 25 mg TMRE [Tetramethylrhodamine ethyl ester]

22221 25 mg TMRM [Tetramethylrhodamine methyl ester]

Mito

chon

dria

l Mem

bran

e Po

tent

ial

Cell Meter™ Mitochondrial Membrane Potential Assay Kit *Orange Fluorescence Optimized for Flow Cytometry*


22804 1 kit < - 15 oC

This particular kit is designed to detect cell apoptosis by measur-ing the loss of the mitochondrial membrane potential(MMP). The collapse of mitochondrial membrane potential coincides with the opening of the mitochondrial permeability transition pores, leading to the release of cytochrome C into the cytosol, which in turn trig-gers other downstream events in the apoptotic cascade.

Our Cell Meter™ Orange Mitochondrial Membrane Potential Assay Kit provides all the essential components with an optimized as-say method. This fluorimetric assay uses our proprietary cationic MitoLite™ Orange for the detection of apoptosis in cells with the loss of mitochondrial membrane potential. In normal cells, the red fluorescence intensity is increased when MitoLite™ Orange is accumulated in the mitochondria. However, in apoptotic cells, the fluorescence intensity of MitoLite™ Orange is decreased follow-ing the collapse of MMP. Cells stained with MitoLite™ Orange can be visualized with a flow cytometer at 488 nm excitation with red emission (FL2 channel). The kit can be used together with other reagents, such as Cell Meter™ Phosphatidylserine Apoptosis Assay Kit (22835) for multi-parametric study of cell vitality and apoptosis. The kit is optimized for screening apoptosis activators and inhibitors with a flow cytometer.

Cell Meter™ Mitochondrial Membrane Potential Assay Kit *Orange Fluorescence Optimized for Microplate Reader*


22805 1 kit < - 15 oC

Our Cell Meter™ assay kits are a set of tools for monitoring cellular functions. There are a variety of parameters that can be used. This particular kit is designed to detect cell apoptosis by measuring the loss of the mitochondrial membrane potential (MMP). The collapse of mitochondrial membrane potential coincides with the opening of the mitochondrial permeability transition pores, leading to the release of cytochrome C into the cytosol, which in turn triggers other downstream events in the apoptotic cascade.

This fluorimetric assay uses our proprietary cationic MitoLite™ Orange for the detection of the mitochondrial membrane potential change in cells. In normal cells, the orange fluorescence intensity is increased when MitoLite™ Orange is accumulated in the mito-chondria. However, in apoptotic cells, the fluorescence intensity of MitoLite™ Orange is decreased following the collapse of MMP. Cells stained with MitoLite™ Orange can be fluorometrically monitored at Ex/Em = 540/590 nm. Our Cell Meter™ Orange Mitochondrial Mem-brane Potential Assay Kit provides all the essential components with an optimized assay method. The kit can be used for screening acti-vators and inhibitors of apoptosis. And the assay can be performed in a convenient 96-well and 384-well fluorescence microtiter-plate format without a wash step.

Figure 7.43. The decrease in fluorescence intensity of MitoLite™ Orange with the addition of FCCP in Jurkat cells. Jurkat cells were loaded with MitoLite™ Orange alone (Blue) or in the presence of 30 μM FCCP (Red) for 15 minutes. The fluorescence intensity of MitoLite™ Orange was measured with a FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer using FL2 channel.

Figure 7.44. The decrease in the fluorescence intensity of MitoLite™ Orange with the addition of FCCP in HeLa cells. HeLa cells were loaded with MitoLite™ Orange alone or in the presence of 20 μM FCCP for 15 minutes. The fluorescence intensity of MitoLite™ Orange was measured 30 minutes after adding assay buffer with a FlexStation™ microplate reader (Molecular Devices) at Ex/Em = 540/590 nm (cut off 570 nm, bottom read). 7

168737j1_cat.indd 96 2/4/11 11:56 AM




7

Mitochondrial M

embrane Potential

7

Cell Meter™ NIR Mitochondrial Membrane Potential Assay Kit *Optimized for Flow Cytometry*


22802 1 kit < - 15 oC

This particular kit is designed to detect cell apoptosis by measur-ing the loss of the mitochondrial membrane potential (MMP). The collapse of mitochondrial membrane potential coincides with the opening of the mitochondrial permeability transition pores, leading to the release of cytochrome C into the cytosol, which in turn trig-gers other downstream events in the apoptotic cascade.

This fluorometric assay uses our proprietary cationic MitoLite™ NIR for the detection of apoptosis in cells with the loss of mitochondrial membrane potential. In normal cells, the red fluorescence intensity is increased when MitoLite™ NIR is accumulated in the mitochon-dria. However, in apoptotic cells, MitoLite™ NIR stain intensity is de-creased following the collapse of MMP. Cells stained with MitoLite™ NIR can be visualized with flow cytometry at red excitation and far red emission (FL4 channel). Our Cell Meter™ NIR Mitochondrial Membrane Potential Assay Kit provides all the essential compo-nents. The kit can be used together with other reagents, such as blue laser excited propidium iodide and Cell Meter™ Phosphati-dylserine Apoptosis Assay Kit (22790) for studying cell vitality and apoptosis. The kit is optimized for screening apoptosis activators and inhibitors with flow cytometry.

Cell Meter™ NIR Mitochondrial Membrane Potential Assay Kit *Optimized for Microplate Reader Assays*


22803 1 kit < - 15 oC

Our Cell Meter™ assay kits are a set of tools for monitoring cellular functions. There are a variety of parameters that can be used. This particular kit is designed to detect cell apoptosis by measuring the loss of the mitochondrial membrane potential (MMP). The collapse of mitochondrial membrane potential coincides with the opening of the mitochondrial permeability transition pores, leading to the release of cytochrome C into the cytosol, which in turn triggers other downstream events in the apoptotic cascade.

Our Cell Meter™ NIR Mitochondria Membrane Potential Detection Kit provides all the essential components with an optimized assay method. This fluorometric assay uses our proprietary cationic Mito-Lite™ NIR for the detection of the mitochondrial membrane poten-tial change in cells. In normal cells, the red fluorescence intensity is increased when MitoLite™ NIR is accumulated in the mitochondria. However, in apoptotic cells, MitoLite™ NIR stain intensity is de-creased following the collapse of MMP. Cells stained with MitoLite™ NIR can be monitored fluorimetrically at 660-680 nm with excitation at 620-640 nm. The kit can be used for screening apoptosis activa-tors and inhibitors. The assay can be performed in a convenient 96-well and 384-well fluorescence microtiter-plate format.

Figure 7.45. The decrease in fluorescence intensity of MitoLite™ NIR with the addition of FCCP in Jurkat cells. Jurkat cells were loaded with MitoLite™ NIR alone (blue line) or in the presence of 50 μM FCCP (red line) for 10 minutes. The fluorescence intensity of MitoLite™ NIR was measured with a FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer using FL4 channel.

Figure 7.46. The decrease in MitoLite™ NIR fluorescence with the addition of FCCP in HeLa cells. HeLa cells were loaded with MitoLite™ NIR alone or in the presence of 20 μM FCCP for 15 minutes. The fluo-rescence intensity of MitoLite™ NIR was measured 30 minutes after adding assay buffer with a FlexSta-tion™ microplate reader (Molecular Devices) at Ex/Em = 640/680 nm (cut off 665 nm, bottom read).

Related Products





17550 0.5 mL Nuclear Green™ DCS1

17551 0.5 mL Nuclear Orange™ DCS1

17540 0.5 mL Nuclear Green™ LCS1

17541 0.5 mL Nuclear Orange™ LCS1

168737j1_cat.indd 97 2/4/11 11:56 AM




Related Products




15204 25 mg 2',7'-Dichlorodihydrofluorescein diacetate [2',7'-Dichlorofluorescin diacetate]

15203 25 mg Dihydrofluorescein diacetate [Fluorescin diacetate]

15206 10 mg Dihydrorhodamine 123

15207 5x1 mg Dihydrorhodamine 123 *Air-free packaging*

15200 25 mg Hydroethidine [Dihydroethidium]

ROS

Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Red Fluorescence*


22901 1 kit < - 15 oC

Reactive oxygen species (ROS) are natural byproducts of the normal metabolism of oxygen and play important roles in cell signaling. However, during oxidative stress-related states, ROS levels can increase dramatically. The accumulation of ROS results in significant damage to cell structures. The role of oxidative stress in cardiovas-cular disease, diabetes, osteoporosis, stroke, inflammatory diseases, a number of neurodegenerative diseases and cancer has been well established. The ROS measurement will help to determine how oxidative stress modulates varied intracellular pathways. Amplite™ Fluorimetric ROS Assay Kit uses our unique ROS sensor to quantify ROS in live cells. Amplite™ ROS Red is cell-permeable. It generates the red fluorescence when it reacts with ROS. The kit is an optimized “mix and read” assay format.

The Amplite™ Fluorimetric ROS Assay Kit provides a sensitive, one-step fluorimetric assay to detect intracellular ROS in live cells with 1-2 hours incubation. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. Its signal can be easily read using either a fluorescence microplate reader or a fluorescent micro-scope at Ex/Em = 520/605 nm. It can be used to either quantify the ROS activities or screen the ROS inhibitors.

7

Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Green Fluorescence*


22900 1 kit < - 15 oC

Reactive oxygen species (ROS) are natural byproducts of the normal metabolism of oxygen and play important roles in cell signaling. However, during oxidative stress-related states, ROS levels can increase dramatically. The accumulation of ROS results in significant damage to cell structures. The role of oxidative stress in cardiovas-cular disease, diabetes, osteoporosis, stroke, inflammatory diseases, a number of neurodegenerative diseases and cancer has been well established. The ROS measurement will help to determine how oxidative stress modulates varied intracellular pathways. Amplite™ Fluorimetric ROS Assay Kit uses our unique ROS sensor to quantify ROS in live cells. Amplite™ ROS Green is cell-permeable. It gener-ates the green fluorescence when it reacts with ROS. The kit is an optimized “mix and read” assay format that is compatible with HTS liquid handling instruments.

The Amplite™ Fluorimetric ROS Assay Kit provides a sensitive, one-step fluorimetric assay to detect intracellular ROS in live cells with one hour incubation. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. Its signal can be easily read using either a fluorescence microplate reader or a fluorescence microscope at Ex/Em = 490/520 nm.

Figure 7.47. Detection of ROS in Jurkat cells. Jurkat cells were seeded on the same day at 300,000 cells/100 μL/well in a 96-well black wall/clear bottom Costar plate. The ROS assay loading solution(100 μL/well) was added and incubated in a 5% CO2, 37 °C incubator for 1 hour. And then the cells were treated with 1mM, 0.1mM or without H2O2 for 30 minutes.

Figure 7.48. Detection of ROS in Jurkat cells. Jurkat cells were seeded on the same day at 300,000 cells/100 μL/well in a 96-well black wall/clear bottom Costar plate. The ROS assay loading solution (100 μL/well) was added and incubated in a 5% CO2, 37 °C incubator for 1 hour. And then the cells were treated with or without 1mM H2O2 for 2 hours.

168737j1_cat.indd 98 2/4/11 11:56 AM



Reporter Gene A

nalysis

Reporter Gene Analysis 8

168737j1_cat.indd 99 2/4/11 11:56 AM



Repo

rter

Gen

e A

naly

sis

8

reporter gene assay kits at-a-glance*

Reporter Gene Fluorescene Luminescence

Beta-Galactosidase Assay (LacZ) 12601

Firefly Luciferase Reporter Gene 12518, 12519 &12520

Gaussia Luciferase Reporter Gene 12530, 12531 &12532

Renilla Luciferase Reporter Gene 12535, 12536 &12537


168737j1_cat.indd 100 2/4/11 11:56 AM



Related Products


101

www.aatbio.com Reporter Gene AnalysisG

alactosidase/Gaussia Luciferase

Amplite™ Fluorimetric Beta-Galactosidase Assay Kit *Green Fluorescence*


12601 1 kit < - 15 oC

E. coli β-galactosidase is a 464 kD tetramer. Each unit of β-galactosidase consists of five domains, the third of which is the active site. It is an essential enzyme in cells. Deficiencies of this enzyme can result in galactosialidosis or Morquio B syndrome. In E. coli, β-galactosidase is produced by the activation of LacZ operon. Detection of LacZ expression has become routine to the point of detection of as few as 5 copies of β-galactosidase per cell.

This kit uses the fluorogenic fluorescein digalactoside (FDG) galac-tosidase substrate that can sensitively distinguish LacZ+ from LacZ- cells. The non-fluorescent substate generates the strongly fluores-cent fluorescein upon reaction with galactosidase. It can be used either for detecting galactosidase conjugates in ELISA type assay systems or for monitoring LacZ gene expression in cells. FDG used in the kit is not fluorescent. The galactosidase induced cleavage of FDG gives fluorescein that has the spectra of Ex/Em = 490/515 nm, which can be detected with most fluorescence instruments equipped with a FITC filter set. The kit comes with all the essential components with an optimized assay protocol. It can be used with a fluorescence microplate reader, a fluorescence microscope, or a flow cytometer. It might also be used for screening galactosidase inhibitors or inducers.

Amplite™ Gaussia Luciferase Reporter Gene Assay Kits *Bright Glow*


12530 1 plate < - 15 oC

12531 10 plates < - 15 oC

12532 100 plates < - 15 oC

The most versatile reporter gene is the firefly luciferase. Recently there is steadily increasing use of other luciferases, such as Gaussia luciferase since these reporters are smaller and do not require the presence of ATP. The bioluminescent enzyme derived from the marine copepod Gaussia prince is efficiently secreted from mam-malian cells upon expression. Gaussia luciferase is a 20kDa protein which catalyzes coelenterazine oxidation by oxygen to produce light. Our Amplite™ Gaussia Luciferase Reporter Gene Assay Kits use a proprietary luminogenic formulation to quantify luciferase activity in cell medium. The formulation generates a luminescent product that gives strong luminescence upon interaction with Gaussia luciferase. The kits provide all the essential components that are compatible with HTS liquid handling instruments. They have high sensitivity and can be performed in a convenient 96-well and 384-well microtiter-plate format. The “glow-type” signal with a half-life of one hour provides a consistent signal across large number of assay plates. The assay is compatible with standard cell growth media.

Figure 8.1. ß-galactosidase dose response was measured with Amplite™ Fluorimetric beta-Galac-tosidase Assay Kit in a Costar 96-well black solid plate using Gemini fluorescence microplate reader (Molecular Devices). As low as 0.3 mU/well ß-galactosidase can be detected with 30 minutes incuba-tion time.

Related Products



21151 250 µg Coelenterazine cp *UltraPure grade*

21152 250 µg Coelenterazine f *UltraPure grade*



12505 25 mg D-Luciferin, potassium salt *UltraPure Grade*

14001 5 mg FDG [Fluorescein di-beta-D-galactopyranoside]

8

Figure 8.2. Sectreated Gaussia Luciferase culture medium was measured with Amplite™ Gaussia Luciferase Reporter Gene Assay Kit (blue line) and a commercially available Gaussia Luciferase Assay Kit (red line) in a 96-well white plate with a NOVOstar plate reader (BMG Labtech).

168737j1_cat.indd 101 2/4/11 11:56 AM



Related Products


102

Reporter Gene Analysis www.aatbio.comLu

cife

rase

/Ren

illa

Luci

fera

se

Amplite™ Luciferase Reporter Gene Assay Kits *Bright Glow*


12518 1 plate < - 15 oC

12519 10 plates < - 15 oC

12520 100 plates < - 15 oC

The most versatile and common reporter gene is the luciferase of the North American firefly photinus pyralis. The protein requires no posttranslational modification for enzyme activity. It is not even toxic in high concentration (in vivo) and can be used in pro- and eukaryotic cells. These Amplite™ Luciferase Reporter Gene Assay Kits use a proprietary luminogenic formulation to quantify lucifer-ase activity in live cells and cell extracts. The assay is based on firefly luciferase, a monomeric 61 kD enzyme that catalyses a two-step oxidation of luciferin, which yields light at 560 nm. Our formulation generates a luminescent product that gives strong luminescence upon interaction with luciferase. The kits provide all the essential components with our optimized “mix and read” assay protocol that is compatible with HTS liquid handling instruments. They have high sensitivity and can be used for the assays that require low detec-tion limit. The kits have a fast, simple, and homogeneous biolu-minescence assay for studying gene regulation and function. The “glow-type” signal with a half-life of two to four hours provides a consistent signal across large batches of plates. The assay is compat-ible with the use of standard cell growth media.

Amplite™ Renilla Luciferase Reporter Gene Assay Kit *Bright Glow*


12535 1 plate < - 15 oC

12536 10 plates < - 15 oC

12537 100 plates < - 15 oC

Common reporter genes include beta-galactosidase, beta-gluc-uronidase and luciferase. The most versatile reporter gene is the firefly luciferase. Recently there is steadily increasing use of other lu-ciferases, such as Renilla luciferase since these reporters are smaller and do not require the presence of ATP.

Our Amplite™ Renilla Luciferase Reporter Gene Assay Kit is designed to provide a fast and sensitive method to detect the luciferase from sea pansy (Renilla reniformis). It uses a proprietary luminogenic for-mulation to quantify Renilla luciferase activity in cell-based assays. Our formulation generates a luminescent product that gives strong luminescence upon interaction with Renilla luciferase. The kit pro-vides all the essential components. It has high sensitivity and can be performed in a convenient 96-well and 384-well microtiter-plate format. The “glow-type” signal with a half-life of one hour provides a consistent signal across large number of assay plates. The assay is compatible with standard cell growth media. This kit enables the measurement of primary expression or gene expression with wild type and the synthetic hRluc genes .

Figure 8.3. Luciferase dose response was measured with Amplite™ Luciferase Reporter Gene Assay Kit. The kit can detect as low as 0.1pg/well luciferase with 20 minutes to 5 hours incubation time without losing signal intensity. The integration time was 1 sec. The half life is more than 4 hours.

Related Products



21151 250 µg Coelenterazine cp *UltraPure grade*

21152 250 µg Coelenterazine f *UltraPure grade*



21155 250 µg Coelenterazine n *UltraPure grade*

12505 25 mg D-Luciferin, potassium salt *UltraPure Grade*

8

Figure 8.4. Renilla Luciferase was measured with Amplite™ Renilla Luciferase Reporter Gene Assay Kit in a 96-well black solid plate with a NOVOstar plate reader (BMG Labtech). As low as 1pg/mL (0.1pg/well/100uL) Renilla Luciferase was detected with 30 minutes incubation time (n=3).

168737j1_cat.indd 102 2/4/11 11:56 AM



www.aatbio.com Index Index

9Index

168737j1_cat.indd 103 2/4/11 11:56 AM



Index www.aatbio.comIn

dex

9

Keyword Index

Acetylcholine .......................................................................................................21Acetylcholinesterase.....................................................................................37-38Acid Sphingomyelinase.....................................................................................53ADP Assay...............................................................................................................21Aldehyde Quantitation......................................................................................22Alkaline Phosphatase...................................................................................38-40Anti-fading.............................................................................................................57Apoptosis..................................................................................................75, 77-83ATP Assay................................................................................................................23Biotin Quantitation................................................................................................7Biotinylation.............................................................................................................7BSA Conjugation.....................................................................................................8Calcium Assay (Cell-Based).........................................................................83-86Calcium Quantitation.........................................................................................24cAMP..................................................................................................................86-88Caspase.............................................................................................................76-77Caspase 3/7.....................................................................................................75-76Caspase 8................................................................................................................77Caspase 9................................................................................................................78Catalase....................................................................................................................42Cell Cycle.................................................................................................................91Cell Cytotoxicity....................................................................................................92Cell Tracking....................................................................................................65-66Cell Viability.....................................................................................................89-90Chloride Channel.................................................................................................93Cholesterol Quantitation..................................................................................24Choline Quantitation.........................................................................................25Cell Labeling (Dead Cells)...........................................................................58-61Cell Labeling (Live Cells)..............................................................................62-64Conjugation..............................................................................................................8 BSA.....................................................................................................................8 KLH.....................................................................................................................8Ethanol Quantitation..........................................................................................25F-Actin Labeling.............................................................................................67-68Galactosidase......................................................................................................101Gaussia Luciferase.............................................................................................101Glucose Oxidase...................................................................................................42Glucose Quantitation.........................................................................................26Glutamate Oxidase.............................................................................................43Glutamic Acid.......................................................................................................26Glutathione Assay........................................................................................27, 78GSH....................................................................................................................27, 78GSH/GSSG Ratio...................................................................................................27HDAC........................................................................................................................43Histone Deacetylase...........................................................................................43Hydrogen Peroxide (H2O2)................................................................................93iFluor™ 488 Protein Labeling...........................................................................13iFluor™ 555 Protein Labeling...........................................................................13iFluor™ 594 Protein Labeling...........................................................................14iFluor™ 647 Protein Labeling...........................................................................14iFluor™ 680 Protein Labeling...........................................................................15iFluor™ 700 Protein Labeling...........................................................................15iFluor™ 750 Protein Labeling ..........................................................................16iFluor™ 780 Protein Labeling ..........................................................................16IgG-HRP Conjugate ELISA.................................................................................50Keyhole Limpet Hemocyanin............................................................................8Kinase.......................................................................................................................44KLH Conjugation....................................................................................................8Luciferase ............................................................................................................102 Firefly ...........................................................................................................102 Gaussia.........................................................................................................101 Renilla...........................................................................................................102Lysosomal Staining.......................................................................................69-70Lysyl Oxidase.........................................................................................................44

Maleimide Quantitation......................................................................................9Matrix Mettalloproteinases.......................................................................45-46MDR Assay .............................................................................................................94Membrane Potential...........................................................................................94MESG Phosphate Assay......................................................................................33mFluor™ Violet 420 Protein Labeling............................................................17mFluor™ Violet 450 Protein Labeling............................................................17mFluor™ Violet 520 Protein Labeling............................................................18mFluor™ Violet 560 Protein Labeling............................................................18Mitochondrial Membrane Potential.......................................................95-97Mitochondrial Staining................................................................................71-72MMP Activity...................................................................................................45-46MMP-3 Activity......................................................................................................45Monoamine Oxidase..........................................................................................45Multidrug Resistance.........................................................................................94Myelopeoxidase...................................................................................................47NAD/NADH Assay..........................................................................................29-30NADH Assay...........................................................................................................30NADP/NADPH Assay.....................................................................................31-32NADPH Assay.........................................................................................................32NADPH Regenerating.........................................................................................47Nuclear Apoptosis Assay...................................................................................79Peroxidase........................................................................................................48-49Phosphate Assay...........................................................................................33, 34Phosphatase Assay.......................................................................................38-40Phosphatidylserilne......................................................................................79-83Protease.............................................................................................................51-52 Generic...........................................................................................................51 MMP..........................................................................................................45-46 Renin...............................................................................................................52Proteaseome 20S.................................................................................................51Protein Quantitation...........................................................................................10Protein Labeling......................................................................................................7 Biotin.................................................................................................................7 iFluor.........................................................................................................13-16 mFluor......................................................................................................17-18Pyrophosphate Assay.........................................................................................34Reactive Oxygen Species..................................................................................98Renilla Luciferase...............................................................................................102Renin........................................................................................................................52Reporter Gene Assay........................................................................................102 Firefly Luciferase.......................................................................................102 Galactosidase............................................................................................101 Gaussia Luciferse......................................................................................101 Renilla Luciferase.....................................................................................102ROS...........................................................................................................................98Sphingomyelinase..............................................................................................53Superoxide Dismutase (SOD)..........................................................................54Thiol Quantitation...............................................................................................10Xanthine Oxidase................................................................................................54

168737j1_cat.indd 104 2/4/11 11:56 AM

Index www.aatbio.com

9

Index

Tel:800-990-8053 • Fax: [email protected] • [email protected]


Alphabetical Index

PRODUCT NAME PAGEAmplite™ Calcium Quantitation Kit 24

Amplite™ Cholesterol Quantitation Kit 24

Amplite™ Choline Quantitation Kit 25

Amplite™ Colorimetric Acetylcholinesterase Assay Kit 37

Amplite™ Colorimetric Aldehyde Quantitation Kit 22

Amplite™ Colorimetric Alkaline Phosphatase Assay Kit 38

Amplite™ Colorimetric Biotin Quantitation Kit 7

Amplite™ Colorimetric Maleimide Quantitation Kit 9

Amplite™ Colorimetric NAD/NADH Assay Kit 29

Amplite™ Colorimetric NADP/NADPH Assay Kit 31

Amplite™ Colorimetric Peroxidase Assay Kit 48

Amplite™ Colorimetric Sphingomyelinase Assay Kit 53

Amplite™ Colorimetric Superoxide Dismutase (SOD) Assay Kit 54

Amplite™ Ethanol Quantitation Kit 25

Amplite™ Fluorimetric Acetylcholine Assay Kit 21

Amplite™ Fluorimetric Acetylcholinesterase Assay Kit 37-38

Amplite™ Fluorimetric Aldehyde Quantitation Kit 22

Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit 39-40

Amplite™ Fluorimetric Beta-Galactosidase Assay Kit 101

Amplite™ Fluorimetric Caspase 3/7 Assay Kit 41

Amplite™ Fluorimetric Catalase Assay Kit 42

Amplite™ Fluorimetric Fluorescamine Protein Quantitation Kit 10

Amplite™ Fluorimetric Glucose Oxidase Assay Kit 42

Amplite™ Fluorimetric Glutamate Oxidase Assay Kit 43

Amplite™ Fluorimetric Glutamic Acid Assay Kit 26

Amplite™ Fluorimetric Glutathione Assay Kit 27

Amplite™ Fluorimetric Goat Anti-Mouse IgG-HRP Conjugate ELISA Assay Kit 50

Amplite™ Fluorimetric Goat Anti-Rabbit IgG-HRP Conjugate ELISA Assay Kit 50

Amplite™ Fluorimetric HDAC Activity Assay Kit 43

Amplite™ Fluorimetric Hydrogen Peroxide Assay Kit 28

Amplite™ Fluorimetric Lysyl Oxidase Assay Kit 44

Amplite™ Fluorimetric Maleimide Quantitation Kit 9

Amplite™ Fluorimetric Monoamine Oxidase Assay Kit 45

Amplite™ Fluorimetric Myelopeoxidase Assay Kit 47

Amplite™ Fluorimetric NAD/NADH Assay Kit 29-30

Amplite™ Fluorimetric NADP/NADPH Assay Kit 31-32

Amplite™ Fluorimetric Peroxidase Assay Kit 48-49

PRODUCT NAME PAGEAmplite™ Fluorimetric Proteasome 20S Activity Assay Kit 51

Amplite™ Fluorimetric Renin Assay Kit 52

Amplite™ Fluorimetric Sphingomyelinase Assay Kit 53

Amplite™ Fluorimetric Thiol Quantitation Assay Kit 10

Amplite™ Fluorimetric Xanthine Oxidase Assay Kit 54

Amplite™ Gaussia Luciferase Reporter Gene Assay Kit 101

Amplite™ Glucose Quantitation Kit 26

Amplite™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit 93

Amplite™ Luciferase Reporter Gene Assay Kit 102

Amplite™ Luminometric Alkaline Phosphatase Assay Kit 40

Amplite™ Luminometric Peroxidase Assay Kit 49

Amplite™ MMP-3 Activity Assay Kit 45

Amplite™ Renilla Luciferase Reporter Gene Assay Kit 102

Amplite™ Universal Fluorimetric Kinase Assay Kit 44

Amplite™ Universal Fluorimetric MMP Activity Assay Kit 46

Amplite™ Universal Fluorimetric Protease Activity Assay Kit 51-52

Cell Explorer™ Cell Tracking Kit 65-66

Cell Explorer™ Fixable Dead Cell Labeling Kit 58-61

Cell Explorer™ Live Cell Labeling Kit 62-64

Cell Meter™ Caspase Activity Apoptosis Assay Kit 75, 77-78

Cell Meter™ Cell Viability Assay Kit 89-90

Cell Meter™ Colorimetric Cell Cytotoxicity Assay Kit 92

Cell Meter™ Fluorimetric Cell Cycle Assay Kit 91

Cell Meter™ Fluorimetric Cell Cytotoxicity Assay Kit 92

Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit 98

Cell Meter™ Fluorometric Caspase 3/7 Activity Assay Kit 76

Cell Meter™ Generic Fluorometric Caspase Activity Assay Kit 76-77

Cell Meter™ Intracellular GSH Assay Kit 78

Cell Meter™ JC-10 Mitochondrial Membrane Potential Assay Kit 95

Cell Meter™ Mitochondrial Membrane Potential Assay Kit 96

Cell Meter™ NIR Mitochondria Membrane Potential Assay Kit 97

Cell Meter™ Nuclear Apoptosis Assay Kit 79

Cell Meter™ Phosphatidylserine Apoptosis Assay Kit 79-83

Cell Navigator™ F-Actin Labeling Kit 67-68

Cell Navigator™ Lysosomal Staining Kit 69-70

Cell Navigator™ Mitochondrial Staining Kit 71-72

FluoroQuest™ Anti-fading Kit 57

www.aatbio.com Index



9

Inde

x

PRODUCT NAME PAGEPhosphoWorks™ Colorimetric MESG Phosphate Assay Kit 33

PhosphoWorks™ Colorimetric Phosphate Assay Kit 33

PhosphoWorks™ Fluorimetric ADP Assay Kit 21

PhosphoWorks™ Fluorimetric Phosphate Assay Kit 34

PhosphoWorks™ Fluorimetric Pyrophosphate Assay Kit 34

PhosphoWorks™ Luminometric ATP Assay Kit *Bright Glow* 23

ReadiLink™ BSA Conjugation Kit *For antibody development* 8

ReadiLink™ iFluor™ 488 Protein Labeling Kit 13








ReadiLink™ KLH Conjugation Kit 8

ReadiLink™ mFluor™ Violet 420 Protein Labeling Kit 17




ReadiLink™ Protein Biotinylation Kit 7

ReadiUse™ NADPH Regenerating Kit 47

Screen Quest™ Colorimetric Chloride Channel Assay Kit 93

Screen Quest™ Colorimetric ELISA cAMP Assay Kit 86

Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit 83

Screen Quest™ Fluo-8 No Wash Calcium Assay Kit 84

Screen Quest™ Fluorimetric ELISA cAMP Assay Kit 87

Screen Quest™ Fluorimetric MDR Assay Kit 94

Screen Quest™ Fluorimetric No Wash cAMP Assay Kit 87

Screen Quest™ FRET No Wash cAMP Assay Kit 88

Screen Quest™ Live Cell cAMP Assay Serive Pack 88

Screen Quest™ Luminometric Calcium Assay Kit 84

Screen Quest™ Membrane Potential Assay Kit 94

Screen Quest™ Ratiometric Fura-2 No Wash Calcium Assay Kit 85

Screen Quest™ Rhod-4 No Wash Calcium Assay Kit 85-86

168737j1_CVR_INDEX.indd 106 2/4/11 12:02 PM

Index www.aatbio.com

9

Index



Catalog Number Index

CAT # PAGE 1100 17

1101 17

1102 17

1105 17

1106 17

1107 17

1110 18

1111 18

1112 18

1114 18

1115 18

1116 18

1224 13

1225 13

1226 13

1227 13

1228 13

1229 13

1230 14

1231 14

1232 14

1235 14

1236 14

1237 14

1240 15

1241 15

1242 15

1245 15

1246 15

1247 15

1250 16

1251 16

1252 16

1255 16

1256 16

1257 16

CAT # PAGE 5501 8

5502 8

5521 7

5522 7

5523 9

5524 10

5525 9

10051 22

10052 22

10054 26

10055 27

10056 27

11100 10

11300 42

11301 47

11302 43

11303 45

11304 54

11305 54

11306 42

11400 37

11401 37

11402 38

11403 21

11501 28

11502 28

11503 93

11540 50

11541 50

11551 48

11552 49

11553 48

11559 49

11950 38

11952 39

11953 39

CAT # PAGE 11954 40

11956 40

12518 102

12519 102

12520 102

12530 101

12531 101

12532 101

12535 102

12536 102

12537 102

12601 101

13456 51

13500 51

13501 52

13502 41

13503 41

13510 46

13511 46

13512 45

13530 52

13601 43

13602 53

13621 53

15255 44

15257 29

15258 29

15259 31

15260 31

15261 30

15262 32

15263 30

15264 32

15265 47

20001 57

20003 57

www.aatbio.com Index



9

Inde

x

108

CAT # PAGE 21609 23

21610 23

21611 34

21655 21

21659 33

21660 34

21665 33

22500 58

22501 60

22502 61

22600 58

22601 59

22602 60

22603 61

22604 59

22606 62

22607 63

22609 64

22614 62

22615 63

22616 64

22620 65

22621 65

22622 66

22623 66

22651 69

22655 69

22656 70

22658 70

22660 67

22661 67

22663 68

22664 68

22666 71

22667 72

22668 72

22673 71

22780 92

CAT # PAGE 22781 92

22785 89

22786 89

22787 90

22789 90

22791 79

22792 80

22793 81

22794 80

22795 75

22796 75

22798 77

22799 78

22800 95

22801 95

22802 97

22803 97

22804 96

22805 96

22810 78

22811 79

22821 76

22822 77

22823 76

22831 82

22832 83

22835 81

22836 82

22841 91

22842 91

22900 98

22901 98

31001 44

36005 94

36006 94

36305 84

36306 84

36307 83

CAT # PAGE 36308 83

36309 83

36314 84

36315 84

36316 84

36320 85

36321 85

36330 86

36331 86

36332 86

36333 85

36334 85

36335 85

36340 94

36341 94

36350 93

36360 24

36370 86

36373 87

36376 87

36377 87

36379 88

36380 88

36382 88

40001 25

40005 26

40006 24

40007 25

Documents

AA T AAT Bioquest - INTERCHIM: Home Fluorimetric Fluorescamine Protein Quantitation Kit ..... ..... 10 Amplite Fluorimetric Thiol Quantitation Assay Kit ..... ..... 10 Section 3 ReadiLink