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1/50To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

Thermo Scientific Pierce

Cell Lysis Technical HandbookFeaturing Cell Lysis Reagents and Detergents

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Table of Contents

Thermo Scientific Pierce Cell Lysis Reagents

Selection Guide 1-2

Introduction to Protein Extraction 3-6

Cell Lysis Methods 4-6

Introduction to Thermo Scientific

Cell Lysis Solutions 7-15

B-PERBacterial Protein Extraction Reagents 8-9

I-PERInsect Cell Protein Extraction Reagent 10

M-PERMammalian Protein Extraction Reagent 11

P-PERPlant Protein Extraction Reagent 12-13

T-PERTissue Protein Extraction Reagents 14

Y-PERYeast Protein Extraction Reagents 14-15

Fusion Protein Purification 16-21

Fusion Protein Purification Kits 16-21

GST Orientation Kit 21

Mammalian and Yeast -Gal Kits 22-23

Cell Fractionation 4-

Cell Fractionation Introduction 24

Mem-PEREukaryotic Membrane 25-2

Protein Extraction Kit

Mitochondria Isolation Kits 27-2

NE-PERNuclear and Cytoplasmic Extraction Kit -31

Cell Surface Protein Isolation Kit

Organelle Enrichment Kits 33-34

DNA Extraction 35

Yeast DNA Extraction Kit 35

Lyse and Go

PCR Reagent 35

Detergents 36-40

Introduction to Detergents 36-37

Properties of Common Detergents 36-37

Surfact-Ampsand Surfact-PakDetergents 38

Specialized Detergents 39

Detergent Removal 40

Protease Inhibitors 41-44

Introduction to Protease Inhibitors 41

HaltProtease Inhibitor Single-Use Cocktails 41-42

Halt Protease Inhibitor Cocktails 43

Halt Phosphatase Inhibitor Cocktails 43

Protein Stabilizing Cocktail 44

PMSF 44

Soybean Trypsin Inhibitor 44

Protein Refolding 45-46

Pierce Protein Refolding Kit 45Inclusion Body Solubilization Reagent 46

Buffers 47

RIPA Buffer 47

SuperSignal Technology is protected by U.S. Patent # 6,432,662.SuperSignal Technology is protected by U.S. Patent # 6,432,662.

B-PER Technology is protected by U.S. patent # 6,174,704.B-PER Technology is protected by U.S. patent # 6,174,704.

U.S. patent pending on Mitochondria Isolation Kit and P-PER Reagent Technology.U.S. patent pending on Mitochondria Isolation Kit and P-PER Reagent Technology.

U.S. patent pending on Krypton Protein Stain Technology.U.S. patent pending on Krypton Protein Stain Technology.

WaringWaringis a trademark of Conair Corporation.is a trademark of Conair Corporation.

TritonTritonis a trademark of Rohm and Haas Company.is a trademark of Rohm and Haas Company.

SonifierSonifieris a trademark of Branson Instruments, Inc.is a trademark of Branson Instruments, Inc.

BiomasherBiomasher is a trademark of Nippi, Incorporated.is a trademark of Nippi, Incorporated.

PolytronPolytronis a trademark of Kinematica AG Company.is a trademark of Kinematica AG Company.

OptiPrepOptiPrepis a trademark of Axis-Shield plc.is a trademark of Axis-Shield plc.

ATCC is a registered trademark of the American Type Culture Collection.ATCC is a registered trademark of the American Type Culture Collection.BrijBrij and Tweenand Tween are trademarks of ICI Americas Inc. Corp.are trademarks of ICI Americas Inc. Corp.

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1. The detergent can be removed by dialysis2. Immunoprecipitation3. Halt Protease Inhibitor Cocktail, Product #s 78425

(EDTA-free) and 78430

4. Samples prepared in Mem-PER Reagent can be dia-lyzed if the buffer contains detergent (e.g., CHAPS),otherwise use Pierce SDS-PAGE Sample Prep Kit(Product # 89888)

5. Slide-A-Lyzer MINI Dialysis Units

6. 2-D Sample Prep for Nuclear Proteins (Product #89863) and 2-D Sample Prep for Membrane Proteins(Product # 89864) were designed using our NE-PERand Mem-PER Reagents.

7. Need to lyse mitochondria first.

Thermo Scientific Pierce Cell Lysis ReagentsSelection Guide

1For more information, or to download product instructions, visit www.thermo.com/pierce

Description Organisms/Samples Dialyze1 Compatibility

B-PER Reagent

78243, 165 ml78248, 500 ml

Gram(-) bacteria, S. aureus, H. pylori, E. colistrainsBL21(D3)> JM109> DH5a >M15, Archaebacteria, nematodesand Acinetobactersp.

Yes Reporter assays, IPs2, Western blot, GST- andhistidine-tag purification

B-PER II Reagent78260, 250 ml(A 2X version of B-PER Reagent)

Gram(-) bacteria, S. aureus, H. pylori, E. colistrains BL21(D3)>JM109> DH5>M15, Archaebacteria, nematodes andAcinetobactersp.

Yes Reporter assays, IPs2

, Western blot, GST- andhistidine-tag purification

B-PER PBS Reagent78266, 500 ml

Gram(-) bacteria, S. aureus, H. pylori, E. colistrains BL21(D3)>JM109> DH5>M15, Archaebacteria, nematodes andAcinetobactersp.

Yes Reporter assays, IPs2, Western blot, GST- andhistidine-tag purification

Y-PER Reagent78991, 200 ml78990, 500 ml

S. cerevisiae, Schizo-saccharomyces pombe, C. albicans,B. subtilis, E. coli, P. pastoris, Strep. avidiniiandAcinetobactersp.

No IPs2, Western blot, -Gal enzyme assays, IEF afterdialysis, GST- and histidine-tag purification

Y-PER Plus Reagent78998, 25 ml78999, 500 ml

Yeast (S. cerevisiae) and Acinetobactersp. Yes GST- and histidine-tag purification, Western blot

M-PER Reagent78503, 25 ml

78501, 250 ml78505, 1 L

Cultured mammalian cells, COS-7, NIH 3T3, Hepa 1-6, 293,CHO, MDA, MB231 and FM2

Yes Luciferase,-Gal (low signal), CAT, kinase assays,ELISAs, immobilized glutathione, Western blot

P-PER Plant ProteinExtraction Reagent89803, Kit

Multiple plant organs (leaf, stem, root, seed and flowers);multiple plant species (Arabidopsis, tobacco, maize,soybeans, peas, spinach, rice and other plant tissues);and fresh, frozen and dehydrated plant tissues

No 1-D and 2-D gel electrophoresis, Western blotting,activity assays and protein affinity purifications*

T-PER Reagent78510, 500 ml

Heart, liver, kidney and brain Yes Luciferase,-Gal, CAT, kinase assays, Western blot,ELISAs, immobilized glutathione

I-PER Reagent89802, 250 ml

Baculovirus-infected insect cells grown in suspension ormonolayer culture

No Western blot, 6xHis-tagged protein purification,protein assays and ion-exchange chromatography

NE-PER Reagent78833

Tissue: calf liver. Cultured cells: epithelial (HeLa), fibroid(COS-7), kidney (NIH 3T3), liver (Hepa 1) and brain (C6)

No (CER)Yes (NER)

EMSA (if using < 3 l or 10%, otherwise dialyze firstin SAL MINIs5), Western blot, reporter assays, IEF(after dialysis to reduce salt concentration) and 2-D6

Mem-PER Reagent89826

Cultured cells: brain (C6), epithelial (HeLa), fibroblasts(NIH 3T3) and yeast (S. cerevisiae)

Yes4 Western blot and 2-D6

Mitochondria Isolation Kit forCultured Cells89874

Mitochondria Isolation Kitfor Tissue89801

Mammalian cells

Heart, liver, kidney and brain

Yes7 Western blot, 2-D Western blots, electrophoresis.Applications include apoptosis, signal transductionand metabolic studies.

Pierce RIPA Buffer89900, 100 ml89901, 250 ml

Cultured mammalian cells and cytoplasmic, membraneand nuclear proteins

Yes Reporter assays, protein assays, immunoassaysand protein purification

Lysosome Enrichment Kit forTissues and Cultured Cells89839

Tissues and cultured cells N/A 2-D/MS, electron microscopy, disease profiling,gene expression, signal transduction, and interac-tion or localization studies

Peroxisome Enrichment Kitfor Tissue89840

Heart, liver, kidney and brain N/A 2-D/MS, electron microscopy, disease profiling,gene expression, signal transduction, and interac-tion or localization studies

Nuclei Enrichment Kit for Tissue89841

Heart, liver, kidney and brain N/A 2-D/MS, electron microscopy, disease profiling,gene expression, signal transduction, and interac-tion or localization studies

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* Although kit works without liquid nitrogen/freeze-grinding, Douncehomogenization, blade-shearing or glass-bead agitation for cell disrup-

tion, it is compatible with these alternative mechanical aids

See patent information on inside front cover.

o order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.2

Protein Assay Compatibility Notes

Pierce BCA Assay and Coomassie Plus Assay Protease inhibitors3may be added to prevent protein degradation. Salts,chelating agents and reducing agents can be added for more efficient lysis.Do not exceed 0.5 M NaCl. Better lysis if cells are frozen in B-PER Reagent.

Pierce BCA Assay and Coomassie Plus Assay afterCompat-AbleProtein Assay Reagent Set(Product # 23215) or dilute two to four times

Protease inhibitors3

may be added to prevent protein degradation. Salts,chelating agents and reducing agents can be added for more efficient lysis.Better lysis if cells are frozen in B-PER Reagent.

Pierce BCA Assay and Coomassie Plus Assay afterCompat-Able Protein Assay Reagent Set(Product # 23215) or dilute two to four times

Protease inhibitors3may be added to prevent protein degradation. Salts,chelating agents and reducing agents can be added for more efficient lysis.Better lysis if cells are frozen in B-PER Reagent.

Pierce BCA Assay Protease inhibitors3may be added to prevent protein degradation. Use at roomtemperature. Double incubation time for use at 4C. Use log-phase cells. Forstationary phase cells, add 0.1 M DTT or 20-50 mM TCEP. Will work with 1 mMEDTA. Does not lyse spores. Cannot use with ion exchange columns.

Pierce BCA Assay and Coomassie Plus Assay Protease inhibitors3may be added to prevent protein degradation. Theaddition of up to 2 M NaCl may result in increased efficiency of lysis andprotein yield.

Pierce BCA Assay and Coomassie Plus Assay Protease inhibitors3may be added to prevent protein degradation. Adding150 mM NaCl results in increased efficiency of lysis and higher protein yield in

some cells lines. A PBS rinse of cells prior to lysis removes contaminants suchas phenol red and increases protein yield.

Pierce BCA Assay, Reducing Agent-Compatible;Not compatible with Bradford, Coomassieor the original Pierce BCA Assay

Kit lyses most plant cells without harsh mechanical lysis aids; extremelyfibrous tissues such as woody stems may require mechanical grinding bydevices not included in this kit.

P-PER Extracts can be quantified using the Pierce BCA Protein Assay Kit,Reducing Agent Compatible (Product # 23250).

Pierce BCA Assay (dilute 1:1) andCoomassie Plus Assay

Protease inhibitors3may be added to prevent protein degradation. Mechanicaldisruption of the tissue is still required. Can also be used for cultured cells.

Pierce BCA Assay Protease inhibitors3may be added to prevent protein degradation.

Pierce BCA Assay and Coomassie Plus Assay(dilute CER Reagent mixture four times)

Protease inhibitors3may be added to prevent protein degradation. Packed cellvol.: 2 x 106HeLa cells = 10 l = 20 mg. Tissue yield (calf liver): 3-4 mg cytoplasmicprotein/100 mg tissue; 1-1.5 mg nuclear protein/100 mg tissue. Cell yield (HeLa):300-400 g cytoplasmic protein/106cells; 40-60 g nuclear protein/106cells.

Positive controls tested: cytoplasmic (-Gal, PKC, Hsp90); nuclear (Oct-1, p53,DNA polymerase).

Pierce BCA Assay and Coomassie Plus Assay;hydrophobic phase needs to be dialyzed first;see instruction book

Protease inhibitors3may be added to prevent protein degradation. Can dialyzeagainst another detergent (e.g., CHAPS). Extraction efficiency is generally> 50% with the cell lines tested (having proteins with up to two transmembranesegments).

Pierce BCA Assay (after lysis) Protease inhibitors may be added to prevent protein degradat ion. Douncing wil lincrease isolation efficiency vs. detergent alone; however, multiple samplescan be processed simultaneously using the reagent-based methods.

Pierce BCA Assay Protease inhibitors3may be added to prevent proteolysis and maintainphosphorylation of proteins.

Coomassie Plus The Better Bradford Assay Kit Protease inhibitors3may be added to prevent proteolysis and maintainphosphorylation of proteins.



TRIM

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5/503For more information, or to download product instructions, visit www.thermo.com/pierce

Introduction toProtein Extraction

Protein purification encompasses total protein extraction from

a sample (lysis), specific enrichment and/or isolation of a particular

protein of interest (affinity purification), and removal of interfering

or contaminating substances (sample preparation or clean-up).

Cell lysis is the first step in cell fractionation and protein purification

and, as such, opens the door to a myriad of biological studies. Many

techniques are available for the disruption of cells, including physi-

cal and detergent-based methods. Historically, physical lysis has

been the method of choice for cell disruption; however, physical

methods often require expensive, cumbersome equipment and

involve protocols that can be difficult to repeat due to variability

in the apparatus (such as loose-fitting compared with tight-fitting

homogenization pestles). In recent years, detergent-based lysis

has become very popular due to ease of use, low cost and efficient

protocols. We offer several detergent-based lysis reagents for

preparing whole and fractionated cell lysates that are faster and

more convenient than traditional methods.

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Figure 1. Lipid bilayer constituting the outer plasma membrane of a cell.

Cell Lysis Using Traditional(Non-detergent) Methods

Several methods, including mechanical disruption, liquidhomogenization, sonication, freeze/thaw cycles and manualgrinding (Table 2), are commonly used to physically lyse cells.

These methods have been reviewed extensively.

1-4

Mechanical Disruption

Mechanical methods rely on the use of rotating blades to grindand disperse large amounts of complex tissue, such as liver ormuscle. The WaringBlender and the PolytronMixer arecommonly used for this purpose. Unlike the Waring Blender,which is similar to a standard household blender, the PolytronMixer draws tissue into a long shaft containing rotating blades.The shafts vary in size to accommodate a wide range of volumes,and can be used with samples as small as 1 ml.

All cells have a plasma membrane, a protein-lipid bilayer that,in animal cells, forms a barrier separating cell contents from

the extracellular environment. Lipids constituting the plasmamembrane are amphipathic, having hydrophilic and hydrophobicmoieties that associate spontaneously to form a closed bimolecularsheet (Figure 1). Membrane proteins are embedded in the lipidbilayer, held in place by one or more domains spanning thehydrophobic core. In addition, peripheral proteins bind the inneror outer surface of the bilayer through interactions with integralmembrane proteins or with polar lipid head groups. The natureof the lipid and protein content varies with cell type.

In animal cells, the plasma membrane is the only barrier separating

cell contents from the environment, but in plants and bacteria theplasma membrane is surrounded by a rigid cell wall. Bacterialcell walls are composed of peptidoglycan. Yeast cell walls arecomposed of two layers of -glucan, the inner layer being insoluble

to alkaline conditions. Both of these are surrounded by an outerglycoprotein layer rich in the carbohydrate mannan. Plant cellwalls consist of multiple layers of cellulose. These types of extra-cellular barrier confer shape and rigidity to the cells. Plant cellwalls are particularly strong, making them very difficult to disruptmechanically or chemically. Until recently, efficient lysis of yeastcells required mechanical disruption using glass beads. Bacterialcell walls are the easiest to break compared to these other cell

types. The lack of an extracellular wall in animal cells makes themrelatively easy to lyse.

Clearly, the technique chosen for the disruption of cells, whetherphysical or detergent-based, must take into consideration theorigin of the cells or tissues being examined and the inherentease or difficulty in disrupting their outer layer(s). In addition,

the method must be compatible with the amount of material tobe processed and the intended downstream applications. Thishandbook discusses both non-detergent and detergent-basedlysis techniques and then introduces Thermo Scientific PierceCell Lysis Solutions.

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Liquid Homogenization

Liquid-based homogenization is the most widely used cell-disruptiontechnique for small volumes and cultured cells. Cells are lysedby forcing the cell or tissue suspension through a narrow space,

thereby shearing the cell membranes. Three different types ofhomogenizers are in common use. A Dounce homogenizer consistsof a round glass pestle that is manually driven into a glass tube.A Potter-Elvehjem homogenizer consists of a manually ormechanically driven pestle coated with PTFE Material and shaped

to fit a rounded or conical vessel. The number of strokes and thespeed at which the strokes are administered influences the effec-

tiveness of Dounce and Potter-Elvehjem homogenization methods.

Both homogenizers can be obtained in a variety of sizes to accom-modate a range of volumes. A French press consists ofa piston that is used to apply high pressure to a sample volumeof 40 to 250 ml, forcing it through a tiny hole in the press. Only

two passes are required for efficient lysis due to the highpressures used with this process. The equipment is expensive,but the French press is often the method of choice for breakingbacterial cells mechanically.

Table 2. Techniques used for the physical disruption of cells.

Lysis Method Apparatus Description

Mechanical Waring BlenderPolytron Mixer

Rotating blades grind anddisperse cells and tissues

LiquidHomogenization

Dounce HomogenizerPotter-ElvehjemHomogenizerFrench Press

Cell or tissue suspensionsare sheared by forcing them

through a narrow space

Sonication Sonicator High frequency sound wavesshear cells

Freeze/Thaw Freezer or dry ice/ethanol

Repeated cycles of freezing andthawing disrupt cells through icecrystal formation

Manual Grinding Mortar and pestle Grinding plant tissue, frozen inliquid nitrogen

Sonication

Sonication is the third class of physical disruption commonly usedto break open cells. The method uses pulsed, high-frequency soundwaves to agitate and lyse cells, bacteria, spores, and finely diced

tissue. The sound waves are delivered using an apparatus witha vibrating probe that is immersed in the liquid cell suspension.Mechanical energy from the probe initiates the formationof microscopic vapor bubbles that form momentarily and implode,causing shock waves to radiate through a sample. To preventexcessive heating, ultrasonic treatment is applied in multipleshort bursts to a sample immersed in an ice bath. Sonication isbest suited for volumes < 100 ml.

Freeze/Thaw

The freeze/thaw method is commonly used to lyse bacterialand mammalian cells. The technique involves freezing a cellsuspension in a dry ice/ethanol bath or freezer and then thawing

the material at room temperature or 37C. This method of lysiscauses cells to swell and ultimately break as ice crystals formduring the freezing process and then contract during thawing.Multiple cycles are necessary for efficient lysis, and the processcan be quite lengthy. However, freeze/thaw has been shown toeffectively release recombinant proteins located in the cytoplasmof bacteria3and is recommended for the lysis of mammalian cells

in some protocols.

4

Mortar and Pestle

Manual grinding is the most common method used to disruptplant cells. Tissue is frozen in liquid nitrogen and then crushedusing a mortar and pestle. Because of the tensile strength of thecellulose and other polysaccharides constituting the cell wall,

this method was the fastest and most efficient way to accessplant proteins and DNA before we offered the P-PER PlantProtein Extraction Kit (Page 12).

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Additives/Facilitators

Various agents can aid the cell disruption process. Cells sus-pended in a hypotonic buffer swell and burst readily by physicalshearing. Adding lysozyme (200 g/ml) (Product # 89833; page 8)digests the polysaccharide component of yeast and bacterialcell walls. Alternatively, processing can be expedited by treatingcells with glass beads to facilitate the crushing of cell walls,which is commonly used with yeast cells. Viscosity of a sample

typically increases during lysis due to the release of nucleic acidmaterial. DNase (25-50 g/ml) (Product # 89835; page 8) can beadded to lysate along with RNase (50 g/ml) to reduce thisproblem. Nuclease treatment is not required for sonicated material

because sonication shears chromosomes. Finally, proteolysis canbe a problem whenever cells are manipulated; therefore, proteaseinhibitors (Halt Protease Inhibitors, Product #s 78425 and 78430;page 42) should be added to all samples undergoing lysis.

Cell Lysis Using Detergents

Detergent cell lysis is a milder and easier alternative to physicaldisruption of cell membranes, although it is often used inconjunction with homogenization and mechanical grindingwith a Polytron Mixer.

Detergents break the lipid barrier surrounding cells by solubilizingproteins and disrupting lipid:lipid, protein:protein and protein:lipidinteractions. Detergents, like lipids, self-associate and bind tohydrophobic surfaces. They are composed of a polar hydrophilichead group and a nonpolar hydrophobic tail and are categorizedby the nature of the head group as either ionic (cationic oranionic), nonionic or zwitterionic. Their behavior depends on theproperties of the head group and tail.

Unfortunately, there is no standard protocol available for selectinga detergent to use for membrane lysis. The ideal detergent willdepend on the intended application. In general, nonionic andzwitterionic detergents are milder and less denaturing than ionicdetergents and are used to solubilize membrane proteins whenit is critical to maintain protein function and/or retain nativeprotein:protein interactions for enzyme assays or immunoassays.CHAPS, a zwitterionic detergent, and the TritonX Brand seriesof nonionic detergents are commonly used for these purposes.In contrast, ionic detergents are strong solubilizing agents and

tend to denature proteins, thereby destroying protein activity and

function. Studies assessing protein levels strictly through gelelectrophoresis and Western blotting typically use SDS to fullydenature protein samples by boiling. There are a few commonlyused ionic detergents that are only mildly denaturing, includingsodium cholate and sodium deoxycholate.

The choice of detergent for cell lysis also depends on sample type.Animal cells, bacteria and yeast all have differing requirementsfor optimal lysis due to the presence or absence of a cell wall.Because of the dense and complex nature of animal tissues, theyrequire both detergent and mechanical lysis. In addition to thechoice of detergent, other important considerations for optimalcell lysis include the buffer, pH, salt concentration and temperature.Consideration should be given to the compatibility of the chosen

detergent with downstream applications. If the detergent usedfor lysis must be removed, then a dialyzable detergent shouldbe selected.

References1. Evans, W.H. (1987). In J.B.C. Findlay and W.H. Evans (Eds.), Biological Membranes:A Practical Approach. IRL Press, Oxford, England, p. 1-25.2. McNamee, M.G. (1989). Biotechniques7, 466-475.3. Johnson, B.H. and Hecht, M.H. (1994). Bio/Technology12, 1357-1360.4. Current Protocols in Molecular Biology (1995). John Wiley and Sons, Inc.

(supplement 29) pp. 9.7.1-9.7.2.

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B-PER Bacterial Protein Extraction Reagent

Yields greatly exceed those obtained using standardsonication methods.

Highlights:

bacterial lysates purifies inclusion bodies to near-homoge-neous levels*

E. colicell lysis by

and shake for 10 minutes. Recover soluble proteins by pelletingcell debris. Purify inclusion bodies*from the pellet using anoptimized procedure

avoiding contamination of the recombinant protein. If necessary,the nonionic detergent can be removed by dialysis

1

*Does not solubilize inclusion bodies. To solubilize inclusion bodies, see page 46.

The first step to purify or characterize a recombinant protein is todisrupt the cell and release the protein. B-PER Bacterial ProteinExtraction Reagent offers a gentle method of bacterial cell lysis,while also providing the most efficient way to extract recombinantprotein from E. coli.

Table 1. General considerations for Thermo Scientific B-PER Reagent.

Three convenient reagent formats:Original B-PER Reagent is formulation is also available for applications requiring an amine-free buffer. B-PER II Reagent is a twice-concentrated (2X) versionof the original reagent, enabling more concentrated extracts oraddition to cells already in solution.

Fresh cells and frozen cells:B-PER Reagent is capable of extract-ing proteins from both fresh and frozen cells. However, the extrac-

tion is typically most effective with frozen cells.

E. colistrains:B-PER Reagents work well for many common bac-terial host strains. They are especially suitable for the commonlyused, protease-defective bacterial expression host BL21 strains. If

the lysis is not efficient for a particular bacterial strain, try freezing

the cells before extraction.Soluble proteins and inclusion bodies:Perform a mini-scaleextraction to determine solubility of recombinant proteins beforeperforming larger-scale extractions and purifications. Recombinantproteins expressed in bacteria often form inclusion bodies, espe-cially when expressed at high levels. B-PER Reagents effectivelyextract and remove soluble proteins from the insoluble inclusionbody pellet. When combined with inclusion body solubilization andprotein refolding, purified recombinant protein may be obtainedfrom the inclusion body pellet.

Lysozyme:For inclusion body purification, add lysozyme to digestcell debris and release the inclusion bodies. Lysozyme digestioncan significantly improve inclusion body protein purity; the

lysozyme will be eliminated during subsequent washing steps inthe protocol.

DNase I:When minimizing the amount of B-PER Reagent usedin an extraction, viscosity caused by DNA may prevent efficientcentrifugation of cell debris. In such cases, add a small amount of

DNase I to clear the solution.Insect cells:B-PER Reagent has been tested for the extraction ofrecombinant proteins from insect cells infected by baculovirus.The amount of the reagent required depends on the confluency of

the infected cells.

Optional/supplemental materials:Protease inhibitors, salts,chelating agents, reducing agents, etc. may be added directly to

the reagent for specific applications.

Figure 3.Comparison ofThermo ScientificB-PER Reagentwith sonication.E. coliexpressingGFP was extractedfive times withB-PER Reagent orPBS/sonication.Each extractionwas analyzed bySDS-PAGE.

Figure 4.Comparison ofThermo ScientificB-PER Reagent

with sonicationfor measurementof GFP. E. coliexpressing GFPwas extracted fivetimes with B-PERReagent or PBS/sonication. Eachextraction wasanalyzed by GFPactivity assay.

Reference1. Dorsey, C.W., et al.(2003). Genetic organization of an Acinetobacter baumanniichromosomal region harbouring genes related to siderophore biosynthesis and transport.Microbiology149, 1227-1238.

Ordering Information

Product # Description Pkg. Size

78248 B-PER Bacterial Protein Extraction Reagent 200 ml

78243 B-PER Bacterial Protein Extraction Reagent 165 ml

89833 Lysozyme 5 g

89835 DNase I 5,000 units

78425 Halt Protease Inhibitor Single-Use Cocktail,EDTA-free (100X)

24 x 100 lmicrotubes

78430 Halt Protease Inhibitor Single-Use Cocktail(100X)Includes 0.5 M EDTA Solution (100X), 2.5 ml


Thermo ScientificB-PER Reagent

Extraction Round

**GFP = Green Fluorescent Protein

Extraction Round

vs.

1 2 3 4 5 Pellet

PBS/sonication

1 2 3 4 5 Pellet

GFP**

Rounds of Extraction

GFPActivity

31 2 5

Pellet

4

300

250

200

150

100

50

0

B-PER ReagentPBS/sonication

Thermo Scientific Cell Lysis Solutions

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B-PER Reagent (in Phosphate Buffer)

Yields exceed those obtained using standard sonication.

Highlights:

original (Tris buffer-based) B-PER Bacterial Protein ExtractionReagent formulation

-ing, biotinylation or immobilization

B-PER Reagent (in Phosphate Buffer)

After recombinant protein is extracted from E. coli, it may be fur-ther purified or functionally analyzed. The original B-PER Reagentuses a Tris buffer (20 mM, pH 7.5) system. For some proteins,however, a phosphate buffer system is preferred. B-PER Reagent(in Phosphate Buffer) is designed to address this need. The per-

formance of B-PER Reagent (in Phosphate Buffer) is comparableto that of the original (Tris buffer-based) B-PER Reagent. No sig-nificant difference was observed between these two buffer-basedB-PER Reagents, indicating that the buffer has no impact on theextraction efficiency (Figure 5).


A. B.

Protein(g/ml)

31 2 5Total

4


31 2 5Total

4

120

100

80

60

40

20

0

RLU

30,000

25,000

20,000

15,000

10,000

5,000

0

B-PERReagent(in Phosphate Buffer)

B-PER Reagent

B-PER Reagent(in Phosphate Buffer)

B-PER Reagent

Figure 5. Comparison of Thermo Scientific B-PER Reagent (in PhosphateBuffer) with original B-PER Reagent.Approximately 1 g of bacterial pellets wasextracted five times with 10 ml of either B-PER Reagent (in Phosphate Buffer)or original B-PER Reagent. B-PER Reagent (in Phosphate Buffer) showed apattern similar to that of the original B-PER Reagent; i.e., most of the proteinswere extracted in the first round. The protein concentration is measured by

(A)the Pierce BCA Assay and the green fluorescent protein (GFP) activity isdetermined by (B)a luminescence spectrometer (Perkin-Elmer LS50).



78266 B-PER Reagent (in Phosphate Buffer) 500 ml

B-PER II Bacterial Protein Extraction Reagent

Provides more efficient recombinant protein extraction withsmaller volumes than that of the original B-PER Reagent.

Highlights:

body purification*

with B-PER II Bacterial Protein Extraction Reagent

*Does not solubilize inclusion bodies. To solubilize inclusion bodies, see page 46.

With small (5 ml) volumes, the yields of both total soluble proteinand recombinant green fluorescent protein (GFP) extracted byB-PER II Reagent are twice as high as the yields produced byoriginal B-PER Reagent at the first round (Figure 6). The yield ofboth total protein and GFP from all five rounds of extraction are

similar. Therefore, B-PER II Reagent is ideal for recombinantprotein extraction when a small volume is required.


A. B.

Protein(g/ml)

31 2 5Total

4


31 2 5Total

4

120

100

80

60

40

20

0

RLU

30,000

25,000

20,000

15,000

10,000

5,000

0

B-PER II ReagentB-PER Reagent

B-PER II ReagentB-PER Reagent

Figure 6. Comparison of Thermo Scientific B-PER II Reagent with originalB-PER Reagent. Approximately 1 g of bacterial pellets expressing recombi-nant GFP was extracted five times with 5 ml of either B-PER II Reagent ororiginal B-PER Reagent. The protein concentration is measured by thePierce BCA Assay, and the GFP activity is determined by a luminescencespectrometer (Perkin-Elmer LS50). A:Soluble proteins extracted in eachround. B:Recombinant GFP extracted in each round. Note that at the firstround of extraction, the yield from B-PER II Reagent is much higher than thatof the original B-PER Reagent.



78260 B-PER II Bacterial Protein Extraction Reagent 250 ml

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I-PER Insect Cell Protein Extraction Reagent

An efficient, gentle reagent that provides maximum extractionof soluble proteins.

Highlights:

maximum extraction of soluble proteins from insect cells

6xHis-tagged protein purification, protein assays andion-exchange chromatography

adherent cultured insect cells

I-PER Insect Cell Protein Extraction Reagent enables gentleextraction of soluble protein from baculovirus-infected insectcells grown in suspension or monolayer culture. The baculovirusinsect cell expression system is an efficient and popular systemfor production of recombinant (eukaryotic) proteins in cell culture.Proteins expressed in baculoviral systems can be used for structuralanalyses, biochemical assays and a variety of other applications.I-PER Reagent maintains functionality of extracted proteins and isdirectly compatible with downstream applications such as proteinassays, Western blotting (Figure 7) and 6xHis-tagged proteinpurification (Figure 8).

STAT 6

Cyclin B1

GFP

Lysate Insoluble Pellet Soluble Fraction

Figure 7. Thermo Scientific I-PER Reagent efficiently extracts recombinantproteins from infected Sf9 cells: human Cyclin B1, mouse STAT 6 and GFP.I-PER Reagent extracts were prepared from infected Sf9 cells. Normalizedamounts of total, insoluble and soluble protein were separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) beforeWestern blot analysis.

kDa

Eluted Fractions

E1

E2

E3

E4

E5

E13

E6

E7

E8

E9

E10

E11

E12

Marker

Load

Flow-Through

Pellet

Wash1

Wash2

215

120

84

60

39

28

Figure 8. Affinity purification of 6xHis Cyclin B1 from Thermo Scientific I-PERReagent extract. Baculovirus-infected Sf9 cells were harvested and lysed withI-PER Reagent. I-PER Reagent cell extract was directly loaded onto a nickel-chelated agarose column and purified. Protein samples were separated bySDS-PAGE, and the gel was stained with Thermo Scientific GelCodeTMBlueStain Reagent (Product # 24590).



89802 I-PER Insect Cell Protein Extraction ReagentI-PER Reagent consists of a proprietary nonionicdetergent, 130 mM NaCl and a microbial growth

250 ml

Related Products

78410 Halt Protease Inhibitor Cocktail MixSufficient reagent to treat 200 ml of sample.

Includes: Protease Inhibitor Cocktail0.5 M EDTA Solution

Kit

2 ml2 ml

78415 Halt Protease Inhibitor Cocktail EDTA-FreeSufficient reagent to treat 100 ml of sample.

1 ml



28372 BupHPhosphate Buffered Slaine PacksEach pack yields 500 ml of 0.1 M phosphate, 0.1 M NaCI,pH 7.0 when dissolved in 500 ml water (20 L total).

40 packs


8/13/2019 1601583 Cell Lys Is


M-PER Mammalian Protein Extraction Reagent

Provides highly efficient total protein extraction from culturedmammalian cells.

Highlights:

compatible with Coomassie (Bradford) and the Pierce BCAProtein Assays or SDS-PAGE1

direct use in immunoprecipitation and other affinity purificationprocedures

with subsequent assay systems

washing in suspension

-galactosidase, chloramphenicol acetyl-transferase (CAT) and other reporter gene activities as well orbetter than competitor products and freeze/thaw methods

This unique detergent dissolves cell membranes, does notdenature protein and is compatible with downstream assays.M-PER Reagent extracts 25% and 20% more protein than freeze/

thaw cycles and sonication, respectively (Figure 1, page 7).

Suitable for cell lysis on all sizes of culture plates

Lysis with M-PER Reagent is so efficient that adherent cells donot need to be scraped from the culture dish, especially importantwhen the cells are grown in small-welled plates, such as 96- or24-well plates.

Total protein was recovered efficiently without scraping the cells

by simply adding an appropriate amount of M-PER Reagent andshaking for 5 minutes as compared with the Supplier P lysis bufferin 100 mm, 60 mm, 6-well, 24-well and 96-well plates. (Data avail-able on our website.) This feature also provides the feasibility forhigh-throughput cell lysis and subsequent screening assays.

Compatible with reporter assays, kinase assays,immunoassays and protein assays

M-PER Reagent is compatible with (A) luciferase, (B) -galactosidaseand (C) CAT assays, three popular gene regulation reporter assays(Figure 9). Compared to lysing with Supplier Ps lysis buffer followedby one freeze/thaw cycle (as suggested by the manufacturer) or

the standard freeze/thaw method, M-PER Reagent yielded more or

equivalent enzyme activities.

Thermo ScientificM-PER Reagent

Supplier P + Freeze/Thaw Cycles Freeze/Thaw Cycles

Exp. 1

A. RenillaLuciferase

Exp.2

RLU

6

5

4

3

2

1

0

Exp.1

B.-galactosidase

Exp. 2 Exp. 3

Absorbancea

t420nm

0.16

0.12

0.06

0.02

0.00

Exp.1

C. CAT

Exp. 2

C

ATRelativeActivity

100

10

1

0

Figure 9. Thermo Scientific M-PER Reagent compatibility with reporter assaysin transiently transfected mammalian cells. Mammalian FM2 cells weretransiently transfected with a reporter construct containing the luciferasegene. The transfected cells were lysed with either M-PER Reagent or SupplierP Lysis Buffer and subjected to luciferase assay. For -galactosidase and CATassays, MDA-MB-231 cells were cotransfected with reporter constructsexpressing-galactosidase and CAT, respectively. The transfected cells werelysed with M-PER Reagent or the freeze/thaw method, and the lysates were

assayed for -galactosidase and CAT activity.Reference1. Banyard, J., et al.(2003). Type XXIII collagen, a new transmembrane collagen identified

in metastatic tumor cells. J. Biol. Chem. 278(23), 20989-20994.



78503 M-PER Mammalian Protein Extraction Reagent 25 ml

78501 M-PER Mammalian Protein Extraction Reagent 250 ml

78505 M-PER Mammalian Protein Extraction Reagent 1 L

8/13/2019 1601583 Cell Lys Is


P-PER Plant Protein Extraction Kit

Lyses plant leaves, stem, root, seed and flower cells withoutliquid nitrogen.

The P-PER Plant Protein Extraction Kit offers a new method forperforming plant cell lysis and subsequent protein extraction. TheP-PER Kit includes an organic lysing reagent and two aqueousreagents which, in conjunction with mild mechanical agitation,effectively extract plant protein.

This gentle extraction procedure (Figure 10) avoids harsh mechani-cal lysis aids, such as a mortar and pestle. Extracts are preparedin just 10 minutes and protein quantitation can be accuratelydetermined with the Pierce BCA Protein Assay Kit, ReducingAgent Compatible (Product # 23250). The P-PER Kit is effective for

extracting protein from a variety of plant tissues. The quantity ofprotein extracted using the kit is equal to or exceeds conventionalextraction methods (i.e., freeze/grinding) and a commercially avail-able plant protein extraction reagent (using equal tissue weight tolysing/extraction volumes) (Figure 11).

8. Recover protein extract (i.e.,lower, aqueous layer).

1. Prepare P-PER

Working Solution.

2. Place tissue sample

between mesh screens.

3. Add P-PER

Working Solution.

4. Massage tohomogenize samplein Working Solution.

5. Wi thdraw the lysate. 6 . Add lysate tocentrifuge tube.

7. Centrifuge to partitionorganic and aqueous layers.

ProteinOrganic layer

Figure 10. Thermo Scientific P-PERPlant Protein Extraction Kitprotocol summary.

Highlights:

seed and flowers); multiple plant species (Arabidopsis, tobacco,maize, soybeans, peas, spinach, rice wheat and other plant

tissues); and fresh, frozen and dehydrated plant tissues

homogenization, blade-shearing or glass-bead agitation for celldisruption; however, the P-PER Kit is compatible with these alter-native mechanical aids (Figure 12)

electrophoresis (Figure 13), Western blotting, activity assays, andprotein affinity purifications

Pierce BCA Protein Assay Kit, Reducing Agent Compatible(Product # 23250)

cheesecloth or Miracloth, unlike homebrews

are functional

200

976643

29

20146

3.6

1 2 3 4 5 6 7 8 9 10 1 2 3 4

0.02

0.016

0.009

Leaf

Panel A Panel B

MW (kD)

Protein extracted (mg)/total tissue weight (mg)

Tobacco

HomeBrew

SupplierS

0.02

0.02

0.01

HomeBrew

SupplierS

0.176

0.09

HomeBrew

0.056

0.046

HomeBrew

0.090.0

580.0

63

HomeBrew

SupplierS

Corn Leaves Arabidopsis Soybean Corn Kernel

Seed

ThermoScientific

ThermoScientific

ThermoScientific

ThermoScientific

ThermoScientific

Figure 11. The Thermo Scientific P-PER Kit produces equivalent or higherlevels of extracted protein than traditional and other commercial methods.Fresh leaf tissue from tobacco, maize seedlings and Arabidopsiswere lysed andextracted according to the P-PER Kit protocol, Supplier Ss protocol and aliterature-based (homebrew) protocol. Samples were normalized (weight tissue/volume extract), resolved on a 10% Bis-Tris gel and stained with Imperial

Protein Stain (Product # 24615). Samples were also quantified using the PierceBCA Protein Assay Kit, Reducing Agent Compatible (Product # 23250). Panel A.Lane 1.Molecular weight standards, Lanes 2-4.tobacco leaves, Lanes 5-7.cornleaves and Lanes 8-10. Arabidopsis. Panel B. Lanes 1-2.dehydrated soybeanseed and Lanes 3-4.dehydrated corn kernel. Note:The Supplier S method isrecommended for leaf tissue only. The extracted protein levels and the ratios ofextracted protein per total plant tissue weight were determined for all samples.


8/13/2019 1601583 Cell Lys Is


P-PER Plant Protein Extraction Kit (cont.)

200

976643

29

20146

3.6

0.02

1 2 3 4 5 6 7

0.02 0.02 0.02 0.02 0.02

MW (kD)

Protein extracted (mg)/

total tissue weight (mg)

1. Molecular Weight Marker

2. Mesh Bags

3. Glass Dounce Homogenizer

4. Liquid Nitrogen/Mortar & Pestle

5. Polytron Tissue Grinder

6. BioMasher Device (Cartagen)

7. Polypropylene Pestle (Kontes)

Lanes:

Figure 12. Thermo Scientific P-PER Reagent is compatible with commonmechanical grinding aids. Fresh tobacco leaf tissue was extracted with P-PERReagent Working Solution using common plant tissue grinding aids. Sampleswere normalized (weight tissue/volume extract), resolved on a 4-12% Bis-Trisgel and stained with Imperial Protein Stain (Product # 24615). Samples were

also quantified using the BCA Protein Assay Kit, Reducing Agent Compatible(Product # 23250). Lane 1.Molecular weight marker, Lane 2.mesh bag, Lane 3.Wheaton glass Dounce homogenizer, Lane 4.liquid nitrogen/mortar and pestlegrind, Lane 5.Polytron Tissue Grinder, Lane 6. BioMasherSample Prep Device(Cartegan) and Lane 7.blue polypropylene pestle (Kontes). The extractedprotein levels and the ratios of extracted protein per total plant tissue weightwere determined for all samples.

Figure 13. The Thermo Scientific P-PER Kit is compatible with 2-D gelelectrophoresis.Protein was extracted from 160 mg of Arabidopsisrosetteleaves using the P-PER Kit. Samples were focused on pH 3-10 nonlinear IPGstrips followed by 8-16% SDS-PAGE. (The data was provided by Dr. Sixue Chenat the Donald Danforth Plant Science Center.)



89803 P-PER Plant Protein Extraction KitIncludes: P-PER Reagent A

P-PER Reagent BP-PER Reagent CPolypropylene Mesh Bags

Kit20 ml225 l20 ml20 each

23250 BCA Protein Assay Kit Reducing Agent CompatibleSufficient reagents to perform 250 standardtube assays.Includes: BCA Reagent A

BCA Reagent BCompatibility Reagent

Reconstitution BufferAlbumin Standard (2 mg/ml)

Kit

250 ml25 ml10 x 20 mg

15 ml10 x 1 mlampules

24615 Imperial Protein Stain ReagentSufficient reagent to stain up to 50 mini gels(8 cm x 10 cm).

1 L

24617 Imperial Protein Stain ReagentSufficient reagent to stain up to 150 mini gels(8 cm x 10 cm).

3 x 1 L

Related Products

46628 Krypton Fluorescent Protein Stain (10X)Sufficient reagent to stain four mini gels (8 cm x 10 cm)

20 ml

46629 Krypton Fluorescent Protein Stain (10X)Sufficient reagent to stain 20 mini gels (8 cm x 10 cm)or two to four large-format gels

100 ml

46630 Krypton Fluorescent Protein Stain (10X)Sufficient reagent to stain 100 mini gels (8 cm x 10 cm)or 10 to 20 large-format gels

500 ml

See patent information on inside front cover.

ReferenceShitsukawa, N., et al (2007). Genetic and epigenetic alteration among three homogenousgenes of class E MADS box gene in hexaploid wheat. Plant Cell. 19, 1723-1737.

8/13/2019 1601583 Cell Lys Is


T-PER Tissue Protein Extraction Reagent

Extracts total protein from tissue samples.

Highlights:

(w/v) of tissue to T-PER Reagent, then centrifuge to pelletcell/tissue debris

inhibitors, salts, reducing agents, chelating agents, etc.)1-5

assays, immunoassays, ELISAs, Western blots and/or proteinpurifications1-5

as Pierce Coomassie Plus (Bradford) Protein Assay(Product # 23236)1-5

This reagent uses a proprietary detergent in 25 mM bicine,150 mM sodium chloride (pH 7.6) for tissue cell lysis. The simplecomposition of this reagent provides versatility for many differentapplications. Depending on the application, it may be advanta-geous to add other components, such as protease inhibitors, salts,reducing agents, chelating agents, etc., to the reagent beforeproceeding with the cell lysis. The cell lysate prepared with thisreagent may be used for reporter assays (e.g., luciferase,-galactosidase, chloramphenicol acetyl transferase), proteinkinase assays (e.g., PKA, PKC, tyrosine kinase), immunoassays(e.g., Western blots, ELISAs, RIAs) and/or protein purifications.

Protocol1. Weigh tissue samples. A 1:20 (w/v) ratio of tissue to T-PER

Reagent is optimal for this procedure.

Notes:

a. Protease inhibitors may be added to the T-PER Reagent(if necessary).

b. Smaller volumes of T-PER Reagent may be used if a moreconcentrated protein extract is required.

2. Add the appropriate amount of T-PER Reagent to the tissuesample and homogenize.

3. Centrifuge the sample for 5 minutes to pellet cell/tissue debris.

4. Collect supernatant and continue with downstream analysisor further purification.

References1. Sheng, J.G., et al.(2002). Disruption of corticocortical connections ameliorates amyloid

burden in terminal fields in a transgenic model of Ab amyloidosis. J. Neurosci. 22(22),

9794-9799.2. Jepsen, K.H., et al.(2002). A syndrome of joint laxity and impaired tendon integrity inlumican- and fibromodulin-deficient mice. J. Biol. Chem. 277,35532-35540.

3. Runkuan, Y.,et al.(2002). Lipopolysaccharide induces overexpression of MUC2 andMUC5AC in cultured bililary epithelial cells: possible key phenomenon of heptatolithiasis.Amer. J. Pathol. 161, 1475-1484.

4. Lukashevich, I.S., et al. (2003). Arenavirus-mediated liver pathology: acute lymphocyticchoriomeningitis virus infection of rhesus macaques is characterized by high-level inter-leukin-6 expression and hepatocyte proliferation. J. Virol. 77(3), 1727-1737.

5. Aldred, M.A., et al.(2003). Caveolin-1 and caveolin-2, together with three bone morpho-genetic protein-related genes, man encode novel tumor suppressors down-regulated insporadic follicular thyroid carcinogenesis. Cancer Res. 63, 2864-2871.



78510 T-PER Tissue Protein Extraction Reagent 500 ml

89833 Lysozyme 5 g






Y-PER-Plus Dialyzable Yeast ProteinExtraction Reagent

Highlights:

Protein Assays

Use Y-PER-Plus Reagent to extract functional soluble proteins

and proteins from S. cerevisiaeand P. pastoris(yeast), B. subtilis(gram-positive bacteria), and E. coli(gram-negative bacteria).Protein extraction is achieved by a 20-minute incubation in thereagent at room temperature.



78998 Y-PER-Plus Dialyzable Yeast ProteinExtraction Reagent

25 ml

78999 Y-PER-Plus Dialyzable Yeast ProteinExtraction Reagent

500 ml


8/13/2019 1601583 Cell Lys Is


Y-PER Yeast Protein Extraction Reagent

Easy-to-use solution gently disrupts the tough yeast cell wall inless than 20 minutes at room temperature.

Highlights:

methods (Figure 14)

glass bead lysis (e.g., clinging static-charged beads, protein/bead clumps and runaway beads)

Saccharomyces cerevisiae, Schizosaccharomycespombe, Pichia pastoris andBacillus subtilus

and gram-negative bacteria (Figure 15); suitable for use in adiverse range of situations

Traditionally, protein extraction from yeast required physical dis-

ruption to break through the thick proteinaceous cell envelope;less disruptive lysis methods were possible only with other organ-isms like E. coli. Y-PER Yeast Protein Extraction Reagent was thefirst commercially available yeast lysis reagent to use a mild deter-gent lysis procedure to efficiently release functionally active solu-bilized proteins. Several uses for the Y-PER Reagent have beenoptimized that encompass a broad array of applications rangingfrom fusion-tagged protein purification to microplate-compatibleenzyme assays, and genomic and plasmid DNA extraction fromyeast. Y-PER Reagent has even been used to isolate yeast killervirus double-stranded RNA from killer strains of S. cerevisiae.1

ProtocolThe standard protocol for protein extraction is easy:

1. Add an appropriate volume of Y-PER Reagent to pelletedyeast cells.

2. Incubate at room temperature for approximately 20 minutes.

3. Spin down the debris.

The resulting supernatant is a concentrated protein solution,surpassing typical yields obtained with traditional glass beaddisruption.

Thermo ScientificY-PER Reagent

Glass Beads

S. cerevisiae S. pombe

Protein(g)

B. subtilis

700

600

500

400

300

200

100

0

M M1

S. cerevisiae S. pombe B. subtilis E. coli

2 1 2 1 2 1 2

Figure 14. Thermo Scientific Y-PER Reagent extraction yields greater amountsof usable protein. In all three organisms tested, Y-PER Reagent extracts containmore usable protein than traditional glass bead lysis.

Figure 15. Thermo Scientific Y-PER Yeast Protein Extraction Reagent. Y-PERReagent extraction of protein from two different strains each of S. cerevisiae,S. pombe, B. subtilis and E. coli.The samples were analyzed by 4-20%SDS-PAGE and stained with GelCode Blue Stain Reagent.

Reference1. Liermann, R.T., et al.(2000). BioTechniques28, 64-65.



78990 Y-PER-Yeast Protein Extraction Reagent 500 ml

78991 Y-PER-Yeast Protein Extraction Reagent 200 ml

8/13/2019 1601583 Cell Lys Is


B-PER 6xHis Fusion Protein Column

and Spin Purification KitsOptimized high-capacity purifications.

Highlights of both kits:

lysis achieved with B-PER Reagent

Highlights of the B-PER 6xHis Fusion Protein ColumnPurification Kit:

time (2.5-3 hours)

over-expressed protein per column

Highlights of the B-PER 6xHis Fusion Protein Spin Purification Kit:

fusion protein

The B-PER 6xHis Column and Spin Purification Kits rapidly andefficiently purify 6xHistidine-tagged fusion proteins from bacteriaand from baculovirus-infected insect cells. The fusion proteinis extracted using B-PER Bacterial Protein Extraction Reagentand then purified using a Nickel Chelated Column (Ni-ChelatedColumns) or Spin Column included. The patented detergent in

B-PER Reagent, combined with a small amount of imidazole, effi-ciently removes nonspecifically and/or weakly bound proteins(e.g., proteins rich in histidine residues). The 6xHistidine-taggedproteins are then eluted with excess imidazole (Elution Buffer).

The B-PER 6xHis Fusion Protein Column Kit

The kit protocol produces a high yield of pure 6xHistidine fusionprotein. The column has been tested for loading up to 20 mllysates from 500 ml cultures. However, for optimal results, a 10 mllysate from 250 ml bacterial culture (O.D.600~1.5-3) is suggested as

the starting material. The yield and purity greatly depend on theexpression level and the nature of the recombinant protein. As anexample, we routinely obtain 10-12 mg of 6xHistidine-tagged green

fluorescent protein (GFP) from 250 ml overnight bacterial culturewith more than 90% purity.

B-PER Column Kit (Product # 78100) Example

Using 6xHis-tagged GFP as a model, the purification began withthe extraction of the recombinant protein from bacterial cell pelletsusing B-PER Reagent. Typically, bacterial cell pellets from 250 ml

cultures were resuspended in 10 ml of B-PER Reagent. The resus-pended cells were shaken at room temperature (RT) for 10 minutesto ensure complete cell lysis and maximal soluble protein extrac-tion. The lysate was centrifuged and the supernatant, which con-tained the soluble proteins, was loaded onto a Ni-chelated column.The column was washed twice with 3 ml of 6xHis Wash Buffer 1and three times with 3 ml of 6xHis Wash Buffer 2. The 6xHis WashBuffer 1 and 6xHis Wash Buffer 2 efficiently remove nonspecificproteins from the Ni-chelated column.

The 6xHis-tagged GFP was eluted from the column with the 6xHisElution Buffer (200 mM imidazole). Using this optimized system, 12mg of recombinant GFP was purified from 250 ml of bacterial cul-

ture within 3 hours (Figure 1).

Figure 1. SDS-PAGE analysis of the purification of 6xHis-tagged GFP fromE. coliusing the Thermo Scientific B-PER 6xHis Fusion Protein ColumnPurification Kit. Fractions were collected from each of the purification steps asdescribed in the text and assayed on a gradient 4-20% SDS-polyacrylamide gel.The gel was stained with GelCode Blue Stain Reagent (Product # 24592). Lane1.Crude lysate extracted from E. coliexpressing 6xHis-tagged GFP using B-PERReagent, Lane 2.flow-through of the lysate after loading onto a Ni-chelatedcolumn, Lanes 3-4.two washes with 3 ml of 6xHis Wash Buffer 1, Lanes 5-7.three washes with 3 ml of 6xHis Wash Buffer 2, and Lanes 8-9.6xHis-taggedGFP eluted from the column with 6xHis Elution Buffer. Lane M is the molecularweight marker.

M 1 2 3 4 5 6 7 8 9

Thermo Scientific Fusion ProteinPurification Kits

8/13/2019 1601583 Cell Lys Is


B-PER 6xHis Fusion Protein Column andSpin Purification Kits (cont.)

B-PER Spin Kit (Product # 78300) Example

Using 6xHis-tagged GFP as a model, purification begins withrecombinant protein extraction from bacterial cell pellets viaB-PER Reagent. Bacterial pellets expressing 6xHis-tagged GFPfrom 250 ml cultures were resuspended in 10 ml of B-PER Reagentand shaken for 10 minutes at room temperature (RT) to ensurecomplete cell lysis and maximal soluble protein extraction. Thecellular debris was removed by centrifugation and the superna-

tant, which contained 6xHis-tagged GFP, was incubated with 1 ml(50% bed resin) of nickel-charged resin for 10 minutes with gentleshaking. After collecting the affinity gel by a brief, low-speedcentrifugation, the gel was resuspended in 0.25 ml of 6xHis WashBuffer and transferred to a spin column (0.75 ml per column).The resin was washed once with 0.5 ml of 6xHis Wash Buffer to

remove contamination and the recombinant 6xHis GFP was elutedfour times with 6xHis Elution Buffer (Figure 2). From loading the gelinto the column to eluting recombinant protein, the entire process

takes less than 30 minutes. The yield and purity of 6xHis GFP fromeach elution are shown in Table 1.

Table 1. The yield and purity of four eluents of 6xHis GFP.

Elution 1 2 3 4

Yield (mg) 1.6 0.8 0.25 0.1

Purity (%) 80.7 90.0 95.1 97.8

Figure 2. Purification of 6xHis-tagged GFP using the Thermo Scientific B-PERFusion Protein Spin Kit.Fractions from each purification step were analyzed by4-20% SDS-PAGE and stained with GelCode Blue Stain Reagent. Recombinant6xHis-tagged GFP expressed in E. coliBL21 was first extracted by B-PERReagent (Lane 1). After binding to affinity gel (Lane 2), the 6xHis-tagged GFP-bound gels were transferred to spin columns and washed once with wash buf-fer to remove contamination (Lane 3). The recombinant proteins were elutedfour times to achieve complete elution. Lanes 4-5are eluent 2 and eluent 3 of6xHis-tagged GFP. Lane M is the molecular weight marker.

1 2 3 4 5M



78100 B-PER 6xHis Fusion Protein ColumnPurification KitSufficient reagents for five 6xHis purifications(250 ml per culture).Includes: B-PER Bacterial Protein Extraction Reagent

6xHis Wash Buffer 16xHis Wash Buffer 2Elution BufferNickel Chelated Column

Kit

165 ml45 ml60 ml45 ml5 x 1 ml

78300 B-PER 6xHis Fusion Protein SpinPurification KitIncludes: B-PER Bacterial Protein Extraction Reagent

6xHis Wash BufferElution BufferNickel Chelated Agarose

Spin ColumnsCollection Tubes

Kit

165 ml40 ml45 ml8 ml

16 each80 each

89833 Lysozyme 5 g






Protein Purification Handbook

This 80-page handbook provides protocols

and technical and product informationto help maximize results for affinitypurification procedures. The handbookprovides background, helpful hints and

troubleshooting advice for covalentcoupling of affinity ligands to chroma-

tography supports, avidin:biotin-binding,affinity purification of antibodies, immunoprecipitation and co-immunoprecipitation assays, affinity procedures for contaminantremoval, and related procedures.

Log on to our website or contact your local branch office ordistributor to request a copy.

8/13/2019 1601583 Cell Lys Is


B-PER GST Fusion Protein Column andSpin Purification Kits

Two convenient kits that purify > 10 mg/column or milligramquantities in 30 minutes.


achieved with B-PER Reagent

Highlights of the B-PER GST Fusion Protein ColumnPurification Kit:

commercially available kits

Highlights of the B-PER GST Fusion Protein Spin Purification Kit:

pure 6xHis-tagged fusion protein

The B-PER GST Fusion Protein Column and Spin Purification Kitsare for rapid purification of glutathione S-transferase (GST) fusionprotein from bacteria. Recombinant GST is extracted using theB-PER Bacterial Protein Extraction Reagent, and then purifiedusing Immobilized Glutathione included in each kit. The proprietarydetergent in B-PER Reagent prevents most of thenon-GST protein from binding to the column and efficientlyremoves nonspecifically bound proteins, providing a rapid andefficient method for GST fusion protein purification.

B-PER Column Kit (Product # 78200) Example

The kit components and protocol produce a high yield of pureGST fusion protein. For optimal recovery, a sample size is such

that the expected GST load on the column is 80% of the maximumcapacity (approximately 8 mg GST/column). A 10 ml lysate from250 ml bacterial culture (O.D.600~1.5-3) is optimal.

B-PER Reagent was first used to extract soluble proteins fromEscherichia coliexpressing GST. The extracted lysates were thenloaded onto an immobilized glutathione column. Nonspecificallybound proteins were removed with Wash Buffer 1, which contains50% B-PER Reagent. Wash Buffer 2, which does not containB-PER Reagent, was used to equilibrate the column before theelution of the GST protein from the column. Using the B-PER GSTColumn Purification Kit, 5 mg of GST protein was purified from250 ml of bacterial culture in 2.5 hours (Figure 3). In comparisonwith a leading competitors kit (Figure 4), the B-PER GST ColumnPurification Kit produced four times greater yield withoutcompromising the purity. The higher yield is mainly due to thehigher extraction efficiency of soluble protein.

M 1 2 3 4 5 6

Thermo Scientific Fusion Protein Purification Kits

Figure 3. SDS-PAGE analysis of the GST purification using the ThermoScientific B-PER GST Fusion Protein Column Purification Kit. Fractions fromeach purification step were subjected to SDS-PAGE analysis using a gradient4-20% SDS-polyacrylamide gel and stained with GelCode Blue Stain Reagent.Lane 1.The crude lysate extracted from E. coliwith B-PER Reagent, Lane 2. theflow-through from the crude lysate,

Lanes 3-4. wash fractions of Wash Buffer

1, Lane 5. wash fractions of Wash Buffer 2, and Lane 6: GST eluted from thecolumn with the elution buffer (50 mM glutathione).

Supplier P GST Kit

B-PER GST Column Kit

C

Fraction

W

Absorbanceat260nm

E-3E-2W W E-1

2.5

2.0

1.5

1.0

0.5

0

Figure 4. Comparison of the Thermo Scientific B-PER GST Fusion ProteinColumn Purification Kit vs. a similar kit from Supplier P. The pure protein yieldfrom the B-PER GST Kit is approximately four times higher than that of SupplierP. The W represents the wash steps and the E represents the elution. Notethat most of the GST is in the first elution with the B-PER GST Kit, while themajority of the GST protein is in the second elution with Supplier Ps kit.

8/13/2019 1601583 Cell Lys Is


B-PER GST Fusion Protein Column andSpin Purification Kits (cont.)

The B-PER GST Fusion Protein Spin Purification Kit simplifies theextraction of recombinant proteins. Microspin columns provide

speed, convenience and flexibility for protein research. The kitprovides a highly quick and efficient system for GST fusion proteinpurification.

B-PER Spin Kit (Product # 78400) Example

Bacterial extracts containing GST were incubated with 1.0 ml ofimmobilized glutathione (50% resin) for 10 minutes with gentleshaking. Following centrifugation, the supernatant was discardedand the resin containing bound GST was resuspended in 0.25 mlof GST Wash Buffer. The resuspended resin was then transferred

to a spin column (0.75 ml per spin column) and placed in 2 ml col-lection tubes. Following a brief spin, the GST protein was eluted

four times with 0.5 ml of 25 mM glutathione (reduced) to achievecomplete elution (Figure 5). The GST yield and purity are listed inTable 2.

Table 2. The yield and purity of four elutions of GST-tagged GFP.

Elution 1 2 3 4

Yield (mg) 1.5 0.7 0.3 0.1

Purity (%) 92.0 95.1 97.0 99.1

M 1 2 3 4 5

Figure 5. Purification of GST-tagged GFP using the Thermo Scientific B-PERSpin Kit. Fractions from each purification step were analyzed by SDS-PAGE

and stained with GelCode Blue Stain Reagent. Recombinant GST-tagged GFPexpressed in E. coliBL21 was first extracted by B-PER Reagent (Lane 1).Afterbinding (Lane 2), the GST-tagged GFP-bound resin was transferred to spincolumns and washed once with wash buffer to remove contamination (Lane 3).The recombinant proteins were eluted four times to achieve complete elution.Lanes 4-5are eluant 2 and eluant 3 of GST tagged GFP.



78200 B-PER GST Fusion Protein ColumnPurification KitSufficient reagents for five GST fusionprotein purifications (250 ml per culture).Includes: B-PER Bacterial Protein Extraction Reagent

Immobilized Glutathione ColumnsWash Buffer 1Wash Buffer 2Glutathione

Kit

165 ml

5 x 1 ml60 ml85 ml1 gm

78400 B-PER 6xHis Fusion Protein SpinPurification KitIncludes: B-PER Bacterial Protein Extraction Reagent

Immobilized Glutathione ResinWash BufferGlutathioneSpin ColumnsCollection Tubes

Kit

165 ml8 ml85 ml15 x 16 mg16 each80 each

Electrophoresis and Staining Handbook

The 47-page Electrophoresis andStaining Handbook includes technicaland ordering information for all ThermoScientific Pierce Products you need toseparate your proteins. The well-organized handbook walks you step-by-step

through the electrophoresis process andbeyond.

Log on to our website or contact your local branch office ordistributor to request a copy.

8/13/2019 1601583 Cell Lys Is


Y-PER 6xHis and GST Fusion Protein Column

Purification KitsHigh-capacity purification > 10 mg/column.


short amount of time

Saccharomyces cerevisiae(Figures 6-7), Schizosaccharomyces pombe, Bacillus subtilisand more

Thermo Scientific Fusion ProteinPurification Kits

Figure 6. Purification of a 6xHis-tagged protein from S. cerevisiaeusingThermo Scientific Y-PER Reagent.Strain DY150 carrying plasmid pYIL042C wasgrown and induced with galactose to produce a 6xHis-tagged protein. Inducedcells were harvested at an OD600of 5.3 and lysed using Y-PER Reagent for 20minutes. Extract was applied to a nickel-chelated agarose column and, afterwashing, purified 6xHis-tagged protein (approx. 48 kDa) was eluted using

imidazole. Samples from each purification step were analyzed by SDS-PAGEand stained with Pierce Silver Stain. Lane 1. Lysate, Lane 2. Column flow-through, Lane 3. Wash 1, Lane 4. Wash 2, Lane 5. Elution fraction 1, Lane 6.Elution fraction 2, Lane 7. Elution fraction 3, Lane 8. Elution fraction 4, Lane 9.Elution fraction 5 and Lane 10. Elution fraction 6.

The unicellular nature of yeast, combined with its ability to performeukaryotic post-translational modifications that closely mimicprocesses in higher eukaryotes, has made them important research

tools. The many vectors available, as well as the development oftechniques for working with recombinant expression, have givenyeast an irreplaceable role in the research sector.

1 2 3 4 5 6 7 8 9 10

Y-PER 6xHis Column Kit

The Y-PER 6xHis Fusion Protein Column Purification Kit is for rapidand efficient purification of 6xHis fusion proteins from yeast orbacteria. Protein is extracted using Y-PER Yeast Protein Extraction

Reagent and subsequently purified using one of the pre-packagednickel-chelated columns provided in the kit. The proprietary washbuffers and Y-PER Reagent efficiently removes nonspecificallyand/or weakly bound proteins (e.g., proteins rich in exposedhistidine residues). The 6xHis fusion protein is then eluted from

the column with a buffer that contains a high concentration ofimidazole (Elution Buffer). All contents of this kit are suppliedready to use.

Fresh Cells and Frozen Cells:Y-PER Reagent is capable of extracting proteins equally well fromboth freshly harvested and previously frozen cells.

Cell Density and Strain Variation:Differences in organism, media, strain genotype and growthconditions can have dramatic effects on the yield of cells obtainedfrom a given volume of culture. Following are several suggestionsfor the volume of Y-PER Reagent to add for a given mass of wetcell paste.

Saccharomyces cerevisiae:Y-PER Reagent works equally wellon cells grown to saturation or cells isolated from log-phasegrowth in both rich or synthetic defined media. Use 2.5-5.0 ml ofY-PER Reagent for a 1 g cell pellet, which can be scaled up ordown accordingly.

Schizosaccharomyces pombe:Y-PER Reagent works best oncells grown in media such as Edinburgh Minimal Media (EMM).To achieve adequate lysis of cells grown in rich media such

as YES, they must be harvested during log-phase growth. Use2.5-5.0 ml of Y-PER Reagent for a 1 g cell pellet, which can bescaled up or down accordingly.

Note:For cultures of S. pombegrown past log-phase, increasedincubation temperature (45C) and the addition of proteaseinhibitors to Y-PER Reagent has been shown to increase lysisefficiency.

Bacillus subtilis:Y-PER Reagent will not lyse B. subtilisspores.When using a strain that is able to sporulate, harvest the cellsduring log-phase growth. For strains unable to sporulate, cellscan be grown to saturation prior to lysis. Use 2.5-5.0 ml of Y-PERReagent for a 1 g cell pellet, which can be scaled up or down

accordingly.

Escherichia coli:Y-PER Reagent works very well on E. coli.Use 2.5-5.0 ml of Y-PER Reagent for 1 g cell pellet, which canbe scaled up or down accordingly.

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M 1 2 3 4 5 6 7 8

Figure 7. Purification of GST fromS. cerevisiaeusing Thermo Scientific Y-PER

Reagent.Strain BRS1002 carrying plasmid pYEX4T-2 was grown and inducedwith copper to produce GST. Induced cells were harvested in log-phase andlysed using Y-PER Reagent at room temperature for 20 minutes. Extract wasapplied to an immobilized glutathione column and, after washing, purified GST(27 kDa) was eluted using 10 mM reduced glutathione. Samples from each stepwere analyzed by SDS-PAGE and stained with GelCode Blue Stain Reagent.Lane 1.Lysate, Lane 2.Column flow-through, Lane 3.Wash 1, Lane 4.Wash 2,Lane 5.Elution fraction 1, Lane 6.Elution fraction 2, Lane 7.Elution fraction 3and Lane 8.Elution fraction 4.

Y-PER GST Column Kit

The Y-PER GST Fusion Protein Column Purification Kit is designedfor rapid and efficient purification of glutathione S-transferase(GST) fusion proteins from yeast or bacteria. Expressed GSTfusion proteins is extracted using Y-PER Yeast Protein ExtractionReagent and subsequently purified using one of the ImmobilizedGlutathione Columns provided in the kit. The proprietary washbuffers and Y-PER Reagent efficiently removes nonspecificallyand/or weakly bound proteins. The GST fusion protein is theneluted from the column with a buffer that contains a highconcentration of reduced glutathione.



78994 Y-PER 6xHis Fusion Protein ColumnPurification KitContains sufficient reagents for five 6xHis fusionprotein purifications from Saccharomyces cerevisiae,

Schizosaccharomyces pombe, Bacillus subtilisorEscherichia coli(up to 6 g of wet cell paste/purification).Includes: Y-PER Yeast Protein Extraction Reagent

Nickel Chelated ColumnsWash Buffer 1Wash Buffer 2Elution Buffer

Kit

200 ml5 x 1 ml60 ml60 ml45 ml

78997 Y-PER GST Fusion Protein ColumnPurification KitSufficient reagents for five GST fusion proteinpurifications from Saccharomyces cerevisiae,Schizosaccharomyces pombe, Bacillus subtilisorEscherichia coli(up to 6 g of wet cell paste/purification).Includes: Y-PER Yeast Protein Extraction Reagent

Immobilized Glutathione ColumnsWash Buffer 1Wash Buffer 2Glutathione (reduced)

Kit

200 ml5 x 1 ml60 ml85 ml5 x 184 mg

GST Orientation Kit

Covalently attaches purified GST fusion protein to an affinity matrix.

Highlights:

fusion protein

applications, ELISAs and dot blots

The GST Orientation Kit allows the covalent attachment of a GSTfusion protein to glutathione immobilized on a matrix (crosslinked4% beaded agarose). The affinity matrix can then be used for thepurification of antibodies raised against GST fusion proteins andother ligands that have strong affinity for the GST fusion protein.The purified antibody isolated with the use of this immobilizedantigen support can then be used directly for Western blottingapplications, ELISAs and dot blots.

GlutathioneAgarose GSTProtein

of Interest

Figure 8. GST crosslinked to glutathione.



78201 GST Orientation KitSufficient reagents to prepare 2 x 2 ml affinity columns,

each coupled with 2-15 mg of GST fusion protein anduseful for 10 affinity purifications.Includes: Glutathione (reduced)

Immobilized GlutathioneBupH Modified Dulbeccos PBS Pack(Wash Buffer I)Elution BufferBlocking BufferNeutralization BufferGentle Ag/Ab Elution BufferDisuccinimidyl Suberate (DSS)Resin SeparatorsPorous DiscsColumn ExtendersBupH TBS Pack (Wash Buffer II)

Kit

5 x 184 mg2 x 2 ml500 ml

2 x 15 ml6 ml5 ml100 ml2 x 13.2 mg2521

Y-PER 6xHis and GST Fusion Protein ColumnPurification Kits (cont.)

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Thermo Scientific Mammalian and Yeast-Galactosidase Kits

Mammalian -Galactosidase Assay Kit

One reagent for cell- or lysate-based assays.

Highlights:

Comparison of Thermo Scientific -Gal Assay Kit with

Supplier P -Gal Assay Kit.

Thermo Scientific -Gal Assay Kit Supplier P -Gal Assay Kit

1. Remove media from plate. 1. Remove media from plate.

2. Add-Gal Assay Reagent and incubateat 37C for 30 minutes.

2. Wash cells twice with PBS.

3. Stop is optional. 3. Dilute 5X Reporter Lysis Buffer to 1X.

4. Measure absorbance at 405 nm. 4. Add Reporter Lysis Buffer and

incubate for 15 minutes.

5. Scrape cells.

6. Remove cells to a clean tube, vortexand centrifuge for 2 minutes at 4C for30 minutes.

7. Add 2X Assay Buffer and incubate at37C for 30 minutes.

8. Stop the reactions.

9. Measure absorbance at 405 nm.

4 Steps 9 Steps

Kit Advantages:

1. Our-Gal Assay Kit saves time. The average researcher

preforms a four-step procedure with our system as opposedto a nine-step procedure with the Supplier P system.

2. Our -Gal Assay Reagent contains the active ingredientM-PER Mammalian Protein Extraction Reagent, which allowsextraction of 60% more protein than Supplier Ps cell lysisreagent. More -Gal activity can be detected with our -GalAssay Reagent because cells are lysed efficiently.

Supplier P

-Gal Reagent

2,000

Number of Cells Expressing -Galactosidase

4,000

Absorbanceat405

nm

10,0008,0006,000

2.0

1.5

1.0

0.5

0

Figure 1. Thermo Scientific -Galactosidase Assay Reagent surpasses thecompetition by combining efficient cell lysis with sensitive -Gal assay.Methods: C6 cells expressing -Galactosidase were grown in 96-well plates

and lysed with either M-PER Mammalian Protein Extraction Reagent orSupplier P Cell Lysis Reagent. Either our Mammalian -Gal Assay Reagent orSupplier P 2X Assay Buffer was added to each well, respectively. The reactionswere stopped with 150 l of Stop Solution provided by each kit, and theabsorbance was read at 405 nm using a plate-reading spectrophotometer.



75707 Mammalian-Galactosidase Assay KitIncludes: Mammalian-Galactosidase Assay Reagent

M-PER Mammalian ProteinExtraction Reagent

Stop Solution

Kit25 ml25 ml

25 ml

75710 Mammalian-Galactosidase Assay Reagent 3 x 25 ml

75705 Mammalian-Galactosidase Assay Reagent 25 ml

75706 -Gal Assay Stop Solution 25 ml

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Yeast-Galactosidase Assay Kit

Ideal for identifying protein:protein interactions in vivousingtwo-hybrid systems.

Highlights:

and washing steps (ideal for screening applications)

or quantitative, with no re-streaking involved

The gene encoding -galactosidase (lacZ)of E. colihas beenwidely used as a reporter gene in many different prokaryotic andeukaryotic organisms. In particular, this gene has proven useful forstudying gene expression in the yeast S. cerevisiae.

In addition to its utility in studying the regulation of gene expres-sion, the measurement of -galactosidase activity can be used

to identify protein:protein interactions in vivousing two-hybridsystems. The strength of the interaction is usually verified and/orquantitated using a -galactosidase activity assay.

In contrast to methods using 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal) as a -galactosidase substrate,our reagent system allows for the qualitative or quantitativedetermination of -galactosidase activity in solution directly fromcolonies growing on solid medium. Part of a colony is pickedfrom a plate and resuspended in a mixture of Y-PER Yeast ProteinExtraction Reagent and -galactosidase assay buffer. After a briefincubation period, the solution turns yellow from the hydrolysisof o-nitrophenyl--D-galactopyranoside (ONPG) to o-nitrophenol(ONP) and galactose in a mildly alkaline solution. The assaybecomes quantitative if the quantity of cells in the assay is firstdetermined with an absorbance reading at 660 nm (OD660).

Figure 2. Linearity of -galactosidase assay from cells growing in media ina 96-well plate.Strain BRS1002 carrying plasmid pYX122- -Gal was grownto an OD600of 1.0, then 100 l of cells were transferred to a 96-well plate.At time = 0, 100 l of a 1:1 mixture of lysis reagent and 2X -Gal assay bufferwere added to each well and absorbance at 405 nm was determined. Specificactivity for this sample is 160 units (unit=OD 405x 1,000/time/OD660).

5

Time (minutes)

10

Absorbanceat420nm

3020 2515

0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0

y = 0.0151x + 0.0319R2= 0.9819

Figure 3. Reproducibility of -galactosidase assay using the novel lysisreagent.The average of three serial dilutions from two independent experi-ments performed weeks apart demonstrates the reproducibility of the assay. Inboth experiments, cells were grown to an OD660of 0.7, then 100 l of cells weretransferred to a 96-well plate and serially diluted into fresh media. -galacto-sidase activity was determined after 20 minutes (unit=OD405x 1,000/time/OD660).

In data not shown, good reproducibility and the similar specific activity evendown to 60,000 cells/well resulted with this expression level. Weaker expres-sion may require more cells.



75768 Yeast -galactosidase Assay KitIncludes: Y-PER Yeast Protein Extraction Reagent

2X-galactosidase Assay Buffer1M Na2CO3Stop Solution

Kit25 ml25 ml25 ml

Experiment 1Experiment 2

Cells/well

1 x 106

-galactosidaseAc

tivity

-galactosidaseAct

ivity

5 x 105 2.5 x 105

200

150

100

50

0

Experiment 1Experiment 2

Average of 3 dilutions of cells

200

150

100

50

0

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Cell Fractionation

Cell Fractionation with Detergents

Cell fractionation and protein enrichment are important methodsin the rapidly growing field of proteomics. Isolation of subcellularfractions and concentration of proteins in low abundance allow

for more efficient identification and study of proteins of interest,including the isolation of integral membrane proteins andnuclear proteins.

Membrane ProteinsMembrane proteins constituteapproximately 30% of theeukaryotic proteome1and area key target in drug discoveryresearch. However, they aredifficult to isolate becauseof their hydrophobicity, basicnature and large size.

Triton X-114 is a uniquedetergent that is used to notonly solubilize membrane pro-

teins, but also to separate themfrom hydrophilic proteins via phase partitioning at a physiological

temperature.2Solutions of Triton X-114 are homogeneous at 0C(form a clear micellar solution) but separate into aqueous anddetergent phases above 20C (the cloud point) as micellaraggregates form and the solution turns turbid. With increased

temperature, phase separation proceeds until two clear phasesare formed where proteins partition according to their hydrophilicand hydrophobic features. Membrane proteins are enrichedin the hydrophobic fraction. We use this partitioning method in

the Thermo Scientific Mem-PER Eukaryotic Membrane Protein

Extraction Kit (Product # 89826).3

The protocol involves the gentlelysis of mammalian cells using a mild, proprietary detergentfollowed by membrane protein extraction using the nonionicdetergent Triton X-114. Yeast cells can be fractionated as wellusing the Mem-PER Kit in combination with glass beads.

Compartmentalization of Proteins in Cell Extract Fractions

Nuclear ProteinsDesigned for the stepwiseisolation of nuclear andcytoplasmic proteins using amild detergent, the Thermo

Scientific NE-PER Nuclearand Cytoplasmic ExtractionReagents (Product # 78833)prepare extracts from culturedmammalian cells and tissuesby first disrupting the outercell membrane to obtain thecytoplasmic contents and thenextracting proteins from the

nuclei. Cross-contamination between the two fractions is minimal(10%; Figure 1). With this stepwise fractionation procedure,concentrated nuclear extracts are obtained and gene regulationexperiments are not compromised, as is common when wholecell lysates are analyzed. Prepared extracts are compatible with

many downstream applications, including electrophoretic mobilityshift assays (EMSA) with nuclear extracts, reporter assays withcytosolic extracts, Western blots, enzyme assays, and Pierce BCA

Protein Assays.

Mitochondria Isolation andProtein ExtractionIsolation of mitochondria is

typically a laborious processrequiring single-sampleprocessing with Douncehomogenization. The ThermoScientific MitochondriaIsolation Kit for Cultured

Mammalian Cells (Product #89874) uses a non-mechanical,reagent-based method formitochondria isolation that

allows multiple samples (six) to be processed concurrently.Cultured mammalian cell pellets are gently lysed using aproprietary formulation (patent pending) that results in maximumyield of mitochondria with minimal damage to integrity. Theproduct instructions describe guidelines for optimizing purityvs. yield parameters. The kit also offers an optimized Douncehomogenization procedure, which results in recovery of twice asmuch mitochondria (measured as mitochondrial protein) than thereagent-based method. Both methods use differential centrifugation

to separate the mitochondrial and cytosolic fractions with a

bench-top microcentrifuge and are completed in approximately40 minutes (post-cell harvest). Once isolated, the mitochondriacan be used in downstream applications such as apoptosis, signal

transduction and metabolic studies, as well as to facilitatemitochondrial proteomics efforts.

References1. Wallin, E. and Von Heijne, G. (1998). Protein Sci. 7, 1029-1038.2. Bordier, C. (1981). J Biol. Chem. 256,1604-1607. et al.(2003). Selective enrichment of membrane proteins by partition-

phase separation for proteomic studies. J. Biomed. Biotechnol. 2003(4), 249.Figure 1. The Thermo Scientific NE-PER Reagents stepwise extractionprocess results in minimal cross-contamination between cytoplasmic andnuclear fractions.Oct-1 and hsp90 determined by Western blot analysis. -Galdetermined by activity assay. Typical cross-contamination is 10%.

Cytoplasm

Nucleus

Western Blot

%S

ignal

Western BlotActivity Assay

100

90

80

70

60

50

40

30

20

10

0Oct -1 Nuclear

Localized Protein-Gal CytoplasmicLocalized Protein

hsp90 CytoplasmicLocalized Protein

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27/50To order, call 800-874-3723 or 815-968-0747

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