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
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
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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 H3K4me2 31277
Histone H3K4me3 31278
Histone H3K9me1 31281
Histone H3K9me2 31280
Histone H3K9me3 31279
Histone H3K9ac 31253
Histone H3K14ac 31254
Histone H3K18ac 31273
Histone H3K27ac 31290
Histone H3K27me3 31216
Histone H3K36me2 31218
Histone H3K79me2 31221
Histone H4K20me1 31224
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
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.
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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 H3K27me3 antibody (mAb) 61017
Histone H3K36me2 antibody (mAb) 61019
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
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
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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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Ac
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
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
NFkB p65-conjugated beads 33113
NFkB p50-conjugated beads 33114
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NFκB antibody-conjugated beads
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Raji + wild-type oligo
Raji + mutant oligo
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To learn more about our assays and see a complete listing, please visit us at www.activemotif.com/activity.
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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
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
Description Target Format Cat. No.
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)
HDM inhibitor 5 mg 25 mg
14071 14070
IOX1 HDM inhibitor 5 mg 25 mg
14057 14056
ML-324 HDM inhibitor 5 mg 25 mg
14079 14078
Tranylcypromine hemisulfate
HDM inhibitor 50 mg 250 mg
14047 14046
Description Target Format Cat. No.
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
Description Target Format Cat. No.
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
Description Target Format Cat. No.
BROMODOMAINJQ1 (racemic) Bromodomain
inhibitor5 mg
25 mg14067 14066
Description Target Format Cat. No.
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
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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
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™ 3´UTR 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 3´UTR Reporter GoClone™ Collection includes over 12,000 ready-to-transfect human 3´UTR reporter vectors and 1000 synthetic miRNA target reporters to study miRNA-3´UTR interactions, validate miRNA targets, and measure RNA stability, translation efficiency and the functional impact of miRNAs on a gene-by-gene basis.
-2.5
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No Insert
Interaction of miR-122 with 3´UTR targets.
3´UTR targets of miR-122 were cloned into the LightSwitch 3´UTR 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 3´UTR 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 3´UTR reporters• Synthetic target reporters containing sequences
fully complementary to 1000 human miRNAs• miRNA mimics and inhibitors• Validated 3´UTR controls• Complete screening service to validate 3´UTR
targets for any miRNA
14 www.activemotif.com
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.
100,000
0
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M (log10)
RLU
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
N N
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S
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)
Targeting epigenetic modifying enzymes for drug development has great potential for advancing research in the fields of cancer, inflammation, development and neuro- biology. 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
e-in
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