Epigenetics • Gene Regulation • miRNA

TOOLS FOR DRUG DISCOVERYTARGETING EPIGENETICS & GENE REGULATION

• Epigenetics• Gene Regulation• miRNA

Enabling Epigenetics Research

2 www.activemotif.com

DRUG DISCOVERY TOOLBOXassays, reagents & services to accelerate your research

Epigenetic drug discovery & development programs have attracted much attention and investment in recent years. This is driven by the potential to develop novel therapeutic strategies that can reverse transcriptional and epigenetic abnormalities. Active Motif is the industry leader in providing innovative technologies for epigenetics and gene regulation research. This brochure features assays, reagents & services that can easily be integrated into your research strategy to advance and streamline the drug discovery workflow.

For a complete list of available products, please visit us at www.activemotif.com.

Histone Peptide Array

miRNA Functional Assays

Histone Substrates

Small Molecules

Assay-ready Enzymes

Pathway Analysis

Multiplex Epigenetic Assays

Global Response Assays

Genome-wide Services

Recombinant Nucleosomes

Target-relevant Antibodies

Multiplex Transcription Factor Assays

Screening Tools

TARGET IDENTIFICATION

SCREENING

LEADOPTIMIZATION

PRECLINICAL/CLINICAL TRIALS

DRUGDISCOVERY

[email protected]

COMPREHENSIVE PORTFOLIO OF READY-TO-USE RECOMBINANT PROTEINS

For a complete list of available purified recombinant proteins, please visit us at www.activemotif.com/proteins.

RECOMBINANT PROTEINSassay-ready epigenetic proteins & enzymes

There is no need to waste your valuable time and resources producing proteins for assay development. Active Motif’s comprehensive portfolio of high-quality recombinant proteins, enzymes and domains are ready for use in your specific assays.

With over 300 purified assay-ready recombinant proteins – including transcription factors, highly active DNA and histone modifying enzymes & complexes, and bromo-domains – you are sure to find what you need to develop more efficient assays for your drug discovery and devel-opment programs.

Product Catalog No.

BRD4 (333-460) protein 31446

CARM1 protein 31347

EZH2 protein complex 31337

HDAC6 protein 31346

JARID1B / KDM5B protein 31432

LSD1 / KDM1A protein 31334

NFkB p65 protein 31102

p53 protein 31103

p300 protein 31124

PRC2 Complex 31387

PRMT5 complex 31356

Set2 protein 31358

SIRT1 protein 31340

Smyd3 protein 31407

PROTEINS FOR BIOCHEMICAL ASSAYS

• Enzyme activity assays• Inhibitor screens• Binding studies• FRET• AlphaLISA

OFFERING A COMPREHENSIVE COLLECTION OF RECOMBINANT BROMODOMAINS

• BRDs for BRD4, BRD2, GCN5, SMARCA4 and other relevant targets

• Designed specifically for use in assays and screens

• Methyltransferases• Demethylases• Acetyltransferases• Deacetylases• Chromatin modifiers• Bromodomains

EPIGENETICS

• Hormone receptors• NFkB• JAK/STAT• PPAR• p53• c-Myc

TRANSCRIPTION FACTORS

Inflammatory genes

Transcription

Reduced inflammatory response

I-BET

BRD4

I-BET

AcAc

BRD4AcAc

Inflammatory genes Reduced inflammatory response

Transcription


FULL-LENGTH HISTONESunmodified & modified histone substrates

Active Motif offers an exclusive collection of over 60 full-length unmodified and modified recombinant histones, including site- and degree-specific modifications such as methylation, acetylation and phosphorylation. Because of our patented Expressed Protein Ligation (EPL) and Methylated Lysine Analog (MLA) synthesis technologies, Active Motif is the exclusive source of most of our full-length modified histones.

The outcome of your in vitro biochemical assay is more accurate when the system mimics actual cellular biology. To help you make the correct choice of substrate, Active Motif offers over 60 different recombinant unmodified and modified H1, H2A, H2B, H3 and H4 histones. Use as stand-alone substrates or assemble to generate nucleosomes and oligonucleosomes. Or save time and effort by choosing from our selection of pre-assembled human histone octamers and nucleosomes (see opposite page).

For a complete list of available recombinant histones, please visit us at www.activemotif.com/recombhis.

Product Catalog No.

Histone H3 biotinylated 31271

Histone H2A 31251

Histone H2B 31252

Histone H4 31223

Histone H3K4me1 31208






Histone H3K9ac 31253








Choosing the correct histone substrate for assay development is key to achieving the

best results. Opinion leaders in epigenetics recognize the power of reconstituting

recombinant chromatin for creating biologically relevant substrates.

WHICH HISTONE SUBSTRATE IS RIGHT FOR YOUR ASSAY?

The largest collection of recombinant histones available

• Over 60 recombinant histones• Site- and degree-specific

modifications• Patented synthesis technologies• Biotinylated histones available

for FRET

[email protected]

HISTONE OCTAMERS & NUCLEOSOMESphysiologically relevant assay substrates

Whether performing drug screens, analyz-ing enzyme kinetics or monitoring changes in histone modifications, substrate selection is critical. Pre-assembled Histone Octamers and Nucleosomes better mimic cellular biology when compared to histone proteins alone or synthetic peptides.

In vivo, histones are assembled into octamers and are wound by DNA in chromatin, making octamers and nucleosomes more physiologically relevant substrates than histones and histone derived peptides for in vitro studies. More importantly, some Histone Methyltrans-ferases (HMT), such as DOT1L and NSD enzymes, are significantly more active and specific when using nucleosomal substrates in HMT assays. Nucleosomes are also widely used in HMT screening assays to identify small molecule inhibitors for drug discovery.

Active Motif’s Recombinant Histone Octamers and Nucleosomes come pre-assembled to save you time and money. Histone octamers are comprised of unmodified core histone proteins (H2A, H2B, H3 and H4). For nu-cleosomes, the histone octamer is bound by DNA. Both unlabeled and biotin-labeled versions are available to enable flexibility in your experimental design.

For a complete list of available purified recombinant proteins, please visit us at www.activemotif.com/proteins.

Product Catalog No.

Histone Octamer (H3.1) 31470

Histone Octamer (H3.1) - biotinylated 31471

Histone Octamer (H3.3) 31472

Histone Octamer (H3.3) - biotinylated 31473

Nucleosomes (H3.1) 31466

Nucleosomes (H3.1) - biotinylated 31467

Nucleosomes (H3.3) 31468

Nucleosomes (H3.3) - biotinylated 31469

Nucleosome Preparation Kit 53504

Chromatin Assembly Kit 53500

Nucleosome Assembly Control DNA 53502

ACHIEVE BETTER RESULTS WITH THE RIGHT CHOICE OF SUBSTRATE

ADVANTAGES:

• More physiologically relevant• Improves enzyme activity• Better assay specificity• Biotinylated versions available

1. No enzyme2. No substrate3. Histone Octamers4. Nucleosomes A5. Nucleosomes B

1 2 3 4 5

1 2 3 4 5

NSD1-SET

NSD2-SET

To illustrate the importance of choosing the correct substrate to achieve the best

results, recombinant nucleosomes were compared with histone octamers in a histone

methyltransferase assay using SET domain-containing HMT enzymes MMSET/NSD1-SET

and NSD2-SET. The results show there is greater methyltransferase activity when using

recombinant nucleosomes as substrates when compared to octamers alone.


ANTIBODIESfor discovery & analysis of druggable targets

Active Motif is more than just an antibody supplier. We identify, manufacture and rigorously test each antibody in our vast collection to ensure that we offer only the highest quality product.

We monitor every step from immunogen design and specificity screening to application validation. This thorough process ensures our antibodies meet the high standards for consistency and quality you require to achieve the goals of your drug discovery and development efforts.

• Oncology• Immunology• Inflammation• Neurobiology• Metabolic disorders• Cardiovascular disease

• H2A, H2B, H3 & H4 PTMs• DNA methylation• Bromodomain proteins• Methyltransferases• Demethylases• Acetyltransferases• Deacetylases

EPIGENETICS

• Hormone receptors• NFkB family• STAT family• IRF family• AP-1 family• p53• c-Myc

TRANSCRIPTION FACTORS

• Rigorous application testing• Assay-compatible formulations• Large lot sizes• Monoclonals to histone PTMs

Product Catalog No.

BRD4 antibody (pAb) 39909

DOT1L antibody (pAb) 39953

EED antibody (mAb) 61203

EZH2 antibody (mAb) 39875

HDAC1 antibody (mAb) 39531

HDAC2 antibody (mAb) 39533

Histone H3K4me3 antibody (mAb) 61379



Histone H3K79me2 antibody (pAb) 39143

LSD1 / KDM1A antibody (pAb) 61607

MMSET / WHSC1 antibody (mAb) 39879

Smyd3 antibody (pAb) 61407

For more information and a complete list of available antibodies, please visit us at www.activemotif.com/abs.

OVER 700 KEY DNA METHYLATION, HISTONE AND TRANSCRIPTION FACTOR TARGETS

ANTIBODIES FOR RELEVANT DISEASE AREAS

[email protected]

SCREEN BINDING SPECIFICITYof antibodies, proteins & enzymes

The MODified™ Histone Peptide Array enables rapid, high-throughput screening of antibodies, enzymes and proteins for cross-reactivity or binding interactions with histones and their modifications. Each peptide array contains 384 unique histone modification combinations in duplicate, with up to four separate modifications on the same 19mer peptide.

• Most extensive coverage of modifications on the market – 384 unique combinations enable the study of not only individual sites, but also neighboring effects of modifications on recognition and binding.

• Unique synthesis & conjugation methods – ensure ›95% purity of bound peptides and high peptide den-sity at each spot, which is advantageous for analysis of interaction sites with low binding constants.

• Simple Western blot-like array protocol – no special equipment needed.

Two Array Labeling Kit versions are available:

• MODified Array Labeling Kit – to study antibody and enzyme interactions.

• MODified Protein Domain Binding Kit – to analyze the binding specificity of His-tagged protein reader domains.

Product Catalog No.

MODified™ Histone Peptide Array, 1 array 13001

MODified™ Histone Peptide Array, 5 arrays 13005

MODified™ Array Labeling Kit 13006

MODified™ Protein Domain Binding Kit 13007

Analysis of the MODified Histone Peptide Array using the LI-COR Odyssey Infrared

Imaging System.

The top image shows the binding pattern of the H3K9me3 antibody alone, the middle

image shows the binding pattern of the H3K27me2me3 antibody in combination with

the c-Myc control antibody from the Array Labeling Kit, and the bottom image is the

composite from the Odyssey system. The results of the LI-COR Odyssey Infrared

Imaging system show that dual detection is possible with the MODified Histone Peptide

Arrays.

Table of MODified array reagents recommended for specific applications.

The MODified Array Labeling Kit and the MODified Protein Domain Binding Kit are complementary products containing the buffers and reagents required to process the array for detection.

The above table shows the combination of reagents recommended for each application.

For more information on the MODified Histone Peptide Array and Labeling Kits, please visit us at www.activemotif.com/modified.

Application Array Labeling Kit

Antibody Specificity MODified™ Histone Peptide Array Array Labeling Kit

Enzymatic Effects MODified™ Histone Peptide Array Array Labeling Kit

Protein Domain Specificity MODified™ Histone Peptide Array Protein Domain Binding Kit

Anti-H3K9me3; anti-Rabbit IRDye 800

Anti-H3K27me2me3; anti-Mouse IRDye 680

H3K9me3; H3K27me2me3


MULTIPLEX EPIGENETIC ASSAYSassay specific & off-target effects in a single well

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Streptavidin-phycoerythrin

Biotinylated Histone H3 Ab

Histone containing PTM

PTM Ab-conjugated bead

H3

Me3

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SA-PE

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Partnering with Luminex®, the multiplexingleader, Active Motif has developed the first multiplex epigenetic assay for studying histone post-translational modifications (PTMs). The Histone H3 PTM Multiplex Assay is designed for use with Luminex instruments, enabling high-throughput processing of nanogram-sized samples to simultaneously interrogate multiple PTMs in a single reaction.

Time-consuming Western blots can now be replaced in early target validation programs that correlate global histone modification enrichment or depletion with disease. The Histone H3 PTM Multiplex Assays provide a rapid, highly sensitive, high-throughput method for profiling specific and off-target changes in histone PTMs in a multiplex format. Get more data from less input, in less time and at a lower cost.

ASSAY FEATURES

• Multiplex – the only kit available to perform multiplexed histone modification analysis

• Efficient – use less input & less time than WB to assay multiple histone PTMs

• Sensitive – requires only nanogram amounts of crude acid extracts or purified histones

• High content – simultaneously compare specific and off-target effects in a single reaction

To learn more about our Luminex assays, visit us at www.activemotif.com/luminex.

The Histone H3 PTM Multiplex Assay shows increased histone acetylation in response

to SAHA-mediated HDAC inhibition.

HeLa cells pre-treated with increasing concentrations of the HDAC inhibitor SAHA

were evaluated in a multiplex of H3 pan-acetyl, H3S10ph, H3K9ac, H3K4me3,

H3K27me2 & H3 Total Ab-conjugated beads using the Histone H3 PTM Multiplex Assay.

The results demonstrate the ability of the assay to simultaneously assess specific and

off-target effects of the treatment on histone modification levels. The dashed lines

represent IC50 values determined for the acetyl marks.

Schematic of the Histone H3 PTM Multiplex Assay.

The assay works as a solution-based sandwich ELISA to evaluate histone H3 PTM levels.

SPECIFIC EFFECTS

OFF-TARGET EFFECTS

NO EFFECT

[email protected]

Streptavidin-phycoerythrin

Biotinylated TF consensus binding sequence

Transcription factor (TF)

TF antibody-conjugated bead

Biotin

SA-PE

MULTIPLEX NFkB & AP-1 ASSAYSstudy activity of protein families in multiplex

Traditional methods to assay transcription factor activity, such as Western blot and ELISA, are limited by the inability to quantify DNA binding events or to simultaneously compare activity of members of the same transcription factor family. To overcome these limitations, Active Motif partnered with Luminex®, the industry leader for multiplexing, to develop Transcription Factor Multiplex Assays for studies of AP-1 and NFkB families.

These quantitative, high-throughput/high-content assays are designed as pull-down reactions where lysates are combined with a biotinylated consensus binding sequence for the transcription factor family of interest. Fluorescent-labeled magnetic beads conjugated to anti-bodies specific for each transcription factor, each with a unique signal, are added to capture the analyte(s) of in-terest. Streptavidin-phycoerythrin (SA-PE) is then added to bind biotin. Fluorescent signals from the beads and SA-PE are used to measure the levels of transcription factor-bound DNA with the use of a Luminex instrument.

For more information and a complete list of available products, please visit us at www.activemotif.com/tf-luminex.

Multiplex analysis of NFkB DNA binding activity using the Transcription Factor

Multiplex Kit – NFkB.

The graph shows a multiplex assay in which antibody-conjugated beads for multiple

analytes were combined into a single well for simultaneous comparison of the activity

of various NFkB proteins within the same sample.

Bead-based Transcription Factor Multiplex Assay.

Illustration depicting the design of the Transcription Factor Multiplex assay as a

bead-based pull-down assay for measuring levels of transcription factor-DNA binding

activity.

Product Catalog No.

Transcription Factor Multiplex Kit – AP-1 33100

c-Fos-conjugated beads 33101

FosB-conjugated beads 33102

JunB-conjugated beads 33104

JunD-conjugated beads 33105

Transcription Factor Multiplex Kit – NFkB 33110

RelB-conjugated beads 33111

NFkB p52-conjugated beads 33112



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NFκB antibody-conjugated beads

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Raji + wild-type oligo

Raji + mutant oligo


To learn more about our assays and see a complete listing, please visit us at www.activemotif.com/activity.

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Trichostatin AUntreated

MEASURE GLOBAL CELLULAR RESPONSEtools to assay cellular changes in disease models

Whether you are assaying global changes in transcription factor activity or epigenetic modifications in response to compound treatments, or assessing alterations in regulatory pathways caused by disease, Active Motif offers a variety of high through-put simple-to-use assays to enable you to accurately and efficiently measure such global changes.

Our epigenetic screening assays are robust, reliable and amenable to high-throughput processing, enabling you to quickly and easily profile cellular changes in the activity of a variety of targets.

MEASURE GLOBAL CELLULAR CHANGES

• c-Myc• NFkB• IRF-3• HIF-1• STAT3

• ER• GR• p53• Nrf2• and more...

• H3K27me1• H3K27me3• H3K9ac• H3K14ac• H3S10ph• H3S28ph

TRANSCRIPTION FACTOR BINDING

TransAM® Transcription Factor Assays Assays for 40 different transcription factors, including:

HISTONE MODIFICATIONS

Histone ELISAs for:

FLUORESCENT HDAC ASSAY

• Total Histone• H3K4me1• H3K4me2• H3K4me3• H3K9me2• H3K9me3

EPIGENETIC ENZYME ACTIVITY

• Histone Acetyltransferase (HAT) Assays• Histone Deacetylase (HDAC) Assays• Histone Demethylase (HDM) Assays

[email protected]

For a complete list of available activators and inhibitors, please visit us at www.activemotif.com/smallmol.

SMALL MOLECULESto modulate activity of epigenetic enzymes

Active Motif offers an expanding collection of small molecule compounds (activators and inhibitors) to modulate the activity of proteins that regulate DNA methylation, chromatin and histones.

These compounds are ready for use as epigenetic drug discovery tools for lead generation and assay develop-ment. Our comprehensive selection includes compounds that target DNA Methyltransferases (DNMTs), Histone Acetyltransferases (HATs), Histone Deacetylases (HDACs), Histone Methyltransferases (HMTs), Histone Demethy lases (HDMs), and the BET family of bromodomains. These products can be readily scaled to adapt to the requirements of your research.

Description Target Format Cat. No.

HISTONE DEACETYLASEApicidin HDAC inhibitor 1 mg

5 mg14041 14040

BML-210 HDAC inhibitor 5 mg 25 mg

14049 14048

CUDC-101 HDAC inhibitor 5 mg 25 mg

14061 14060

HPA (Hexyl-4-pentynoic acid)

HDAC inhibitor 10 mg 50 mg

14035 14034

MS-275 HDAC inhibitor 5 mg 25 mg

14043 14042

Panobinostat HDAC inhibitor 5 mg 25 mg

14045 14044

Phenylbutyrate Na HDAC inhibitor 1 g 14033

Romidepsin HDAC inhibitor 1 mg 14083

Trichostatin A HDAC inhibitor 1 mg 5 mg

14039 14038

Tubastatin A HDAC inhibitor 1 mg 5 mg

14085 14084

Valproic acid HDAC inhibitor 5 g 14021

Vorinostat (SAHA) HDAC inhibitor 10 mg 50 mg

14027 14026


LYSINE METHYLTRANSFERASE & LYSINE DEMETHYLASEBIX-01294 HMT inhibitor 5 mg

25 mg14073 14072

Chaetocin HMT inhibitor 200 µg 14051

Daminozide HDM inhibitor 50 mg 250 mg

14059 14058

DMOG HDM inhibitor 10 mg 50 mg

14063 14062

GSK-J1 (cell impermeable)

HDM inhibitor 5 mg 25 mg

14069 14068

GSK-J4 (cell permeable)


14071 14070

IOX1 HDM inhibitor 5 mg 25 mg

14057 14056

ML-324 HDM inhibitor 5 mg 25 mg

14079 14078

Tranylcypromine hemisulfate


14047 14046


SIRTUINAK-7 SIRT2 inhibitor 5 mg

25 mg14055 14054

BML-278 SIRT1 activator 5 mg 25 mg

14025 14024

EX-527 SIRT1 inhibitor 5 mg 25 mg

14029 14028

Piceatannol SIRT1 activator 5 mg 25 mg

14037 14036

Resveratrol SIRT1 activator 100 mg 500 mg

14023 14022

SirtAct SIRT1 activator 5 mg 25 mg

14081 14080

Sirtinol SIRT2 inhibitor 5 mg 25 mg

14075 14074

Splitomycin Sir2p inhibitor 5 mg 25 mg

14087 14086


METHYLATION5-Azacytidine DNMT inhibitor 50 mg

250 mg14103 14102

Decitabine (5-Aza-2́ -deoxycytidine)

DNMT inhibitor 10 mg 50 mg

14101 14100

RG108 DNMT inhibitor 5 mg 25 mg

14105 14104


BROMODOMAINJQ1 (racemic) Bromodomain

inhibitor5 mg

25 mg14067 14066


HISTONE ACETYLTRANSFERASEC646 HAT inhibitor 5 mg

25 mg14053 14052

CTPB HAT activator 5 mg 25 mg

14065 14064

Garcinol HAT inhibitor 10 mg 50 mg

14077 14076


To learn more about our LightSwitch Pathway Screening Assays and Services, please visit us at www.activemotif.com/lightswitch.

In understanding a compound’s mechanism of action it is crucial to identify activation of both intended and unintended cellular pathways. LightSwitch™ Pathway Screening Assays help you characterize cellular responses to compound treatment and understand off-target effects.

Our unique collection of validated promoter reporters measure gene expression changes associated with a number of disease-related biological pathways. Our standard LightSwitch Pathway Screening Assays are available as stable cell lines or transfection-ready plas-mids. Custom pathway sets are also available.

CELL-BASED PATHWAY ANALYSISidentify biological responses to compound treatment

LightSwitch Pathway Screening Services can profile pathway responses to

treatments. The heat map above shows the inducible activity of 29 different reporter

constructs that represent 10 different pathways in response to 15 different treatments.

• Analyze 15 pathways in an HTS environment• Identify compound-mediated pathway activation• Determine the MOA and off-target effects of your

lead compounds• Available as a product or a service

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Cell Treatments

Representative R

eporter Constructs

Tox/Ahr

Controls

log2 ratio treated/untreated

Androgen

CREB

Estrogen

p53

Hypoxia

STAT

Heat shock

NFkB

Glucocorticoid

PATHWAY PROFILING PANEL48 promoters and controls

Pathway activity readout for:

HIF-1a Hypoxia

NFkB Inflammation

CREB cyclic AMP

HSF1 Heat shock

p53 DNA damage, apoptosis

STAT Interferon

SREBP Cholesterol biosynthesis

ER Estrogen

AR Androgen

GR Glucocorticoid

AhR Toxicity

[email protected]

For more complete information on our miRNA targets, mimics, inhibitors and services, please visit www.activemotif.com/ls-3utr.

FUNCTIONAL ASSAYS FOR miRNAsmeasure miRNA function and validate miRNA targets

MicroRNAs (miRNAs) are involved in many areas of disease biology, and are actively being tested in the clinic as drug targets and as therapeutic molecules themselves. The LightSwitch™ 3ÚTR Reporter Assay pro-vides an HTS system that is ideally suited for screening the effects of small molecules and chemical modifications on miRNAs.

The LightSwitch 3ÚTR Reporter GoClone™ Collection includes over 12,000 ready-to-transfect human 3ÚTR reporter vectors and 1000 synthetic miRNA target reporters to study miRNA-3ÚTR interactions, validate miRNA targets, and measure RNA stability, translation efficiency and the functional impact of miRNAs on a gene-by-gene basis.

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Interaction of miR-122 with 3ÚTR targets.

3ÚTR targets of miR-122 were cloned into the LightSwitch 3ÚTR vector and

co-transfected with either miR-122 mimic or a non-targeting control miRNA in K-562

cells. The graph shows the four strongest responders along with two non-responding

controls.

Understanding the biological role of a miRNA often requires identification of downstream targets. The LightSwitch 3ÚTR reporter system is a validated assay that can rapidly identify hundreds of miRNA target genes in a single experiment.

• Measure the effects of small molecules or chemical modifications on miRNA activity

• Validate downstream miRNA targets• Identify all miRNAs that regulate a gene of interest• miRNA screening services also available

PRODUCTS FOR miRNA FUNCTIONAL SCREENS

• 12,000 pre-cloned human 3ÚTR reporters• Synthetic target reporters containing sequences

fully complementary to 1000 human miRNAs• miRNA mimics and inhibitors• Validated 3ÚTR controls• Complete screening service to validate 3ÚTR

targets for any miRNA


REPORTER ASSAYS FOR DRUG SCREENINGaccelerate reporter assay screens with the LightSwitch™ System

The LightSwitch™ Luciferase Assay System dramatically reduces the time investment needed to identify the most responsive promoter to develop as a new HTS reporter assay. Over 18,000 pre-cloned human promoter reporter vectors are available to choose from for immediate incorporation into plate-based screens.

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EquilinEstriolEstroneEE2E2

PROMOTER

Pre-cloned into a Reporter

Vector

3. Measure luciferase activity to determine

functional effects.

RenSPLuciferase

LightSwitch™

Promoter Reporter

GENE OF INTEREST

1. Transfect vector.

2. Treat with

compound.

N N

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For more complete information, please visit us at www.activemotif.com/lightswitch.

Dose response of LightSwitch SYT8 Promoter Reporter construct to estrogen

compounds.

HT1080 cells were co-transfected with a SYT8 Promoter GoClone™ (Product ID

S714388) and an ER cDNA expression plasmid, then treated with five different estrogen

compounds for 24 hours before the luminescence was measured using the LightSwitch

Luciferase Assay Kit.

• 18,000 pre-cloned human promoter reporters for screening expression changes of any human gene

• 100 transcription factor response element reporters for screening transcription factor activity

• Validated promoter reporters for measuring pathway activation are also available (see page 12)

CUSTOM SERVICES AVAILABLE

• Stable cell line generation• Custom assay development• Custom cloning of novel regulatory elements (or

TF response elements)

[email protected]

Targeting epigenetic modifying enzymes for drug development has great potential for advancing research in the fields of cancer, inflammation, development and neurobiology. The in-depth characterization of lead compounds in these areas requires a genome-wide mechanistic perspective of how these compounds alter epigenetic profiles.

Active Motif’s Epigenetic Services team specializes in genome-wide compound characterization and offers this assay as an end-to-end ChIP-Seq service to accelerate your drug discovery and development program.

GENOME-WIDE SOLUTIONScharacterize inhibitors of epigenetic targets

NEW: ChIP-Seq Spike-in Strategy

Traditional ChIP-Seq protocols do not always detect global changes in histone modifications follow-ing treatment with epigenetic inhibitors. Our novel ChIP-Seq spike-in and normalization strategy is able to reveal these differences and is now offered as part of our end-to-end ChIP-Seq Service.• Drosophila chromatin is “spiked in” to a ChIP reaction• A second Drosophila-specific antibody is added to

reliably pull down a small fraction of Drosophila chromatin

• Drosophila tag counts are equalized across samples and used to normalize ChIP-Seq data

To view the full range of services and for more information, please visit us at www.activemotif.com/services.

ChIP-Seq Spike-in Normalization Strategy reveals changes in H3K27me3 levels

following treatment with EZH2 inhibitor compound.

Cells treated with a small molecule inhibitor of EZH2 methyltransferase have dramatic

reductions in global H3K27me3 levels. However, H3K27me3 ChIP-Seq using standard ChIP-

Seq protocols (-) does not detect these differences. Incorporation of Active Motif’s ChIP-Seq

Spike-in Strategy (+) reveals the expected decrease in H3K27me3 ChIP-Seq signal.

RELATED SERVICES

Active Motif also offers several other end-to-end whole-genome services that can be combined to meet your specific research needs.• RNA Pol II ChIP-Seq – measure transcription

rates• RNA-Seq – determine steady-state mRNA levels• MeDIP-Seq – profile genome-wide

DNA methylation

ChIP-Seq Services

PROCESS STEPS

QUALITY CONTROL

Customer fixes cell lines or freezes tissue samples.

1

Chromatin preparation is quantified and sized.

Antibody is ChIP Qualified prior to ChIP-Seq ChIP rxns.

qPCR is used to validate success of the ChIP rxns.

QC by qPCR quantification of yield and library size.

Sequencing to yield ≥ 30 million tags.

Active Motif prepares chromatin and sonicates.

2

Active Motif performs the ChIP reactions.

3

Active Motif constructs ChIP-Seq libraries.

4

Libraries sequenced using Illumina HiSeq.

5

Analysis and Data Delivery.6

• H3K9ac• H3K27me3• H3K4me2

VALIDATED ASSAYS FOR OVER 200 TARGETS

• BRD4• EZH2• LSD1

Spik

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Nor

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www.activemotif.com

Enabling Epigenetics Research

NORTH AMERICA

Toll Free: 877 222 9543Direct: 760 431 1263Fax: 760 431 [email protected][email protected]

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Documents

Epigenetics • Gene Regulation • miRNA