motif vations

THE NEWSLETTER OF ACTIVE MOTIFAPRIL 2016 | VOLUME 17 NUMBER 1

CANCER EPIGENETICS EDITION

Enabling Epigenetics ResearchAntibodies, Kits and Services for Epigenetics & Gene Regulation Research

Epigenetics & Cancer

IN THIS ISSUE
2

Epigenetics & Cancer.

4

RNA Analysis Techniques to Advanceour Understanding of the Transcriptome

5

NEW: Tools to Study Nrf2 Transcription
Factor Activation

6

Practical Guide to Chromatin
Immunoprecipitation Techniques

10 NEW: Determine CRISPR/Cas9
Specificity Using enChIP
11 Quantify Global Changes in 5-mC
& 5-hmC in Normal and Diseased
Samples
12 A Genome-wide 3’UTR Collection for
High-throughput Functional Screeningof miRNA Targets
13 Validated Antibodies Reveal Histonemediated Effects on miRNA in
Non-small Cell Lung Cancer
14 High Quality Bromodomain Proteinsfor Developing Robust Assays for
Epigenetic Drug Discovery

2

Epigenetics, or the heritable changes in gene function that occur
independent of DNA sequence, has become a major focus ofdevelopment and disease research. Countless studies link epigenetic
alterations to developmental processes, including X-chromosome
inactivation, genomic imprinting, axis patterning and differentiation,
as well as to diseases such as cancer, chromosomal instabilities
and mental retardation. Because genetics alone does not provide
an adequate explanation for the complexity of these processes,
a deeper understanding of the role of epigenetics is crucial tounraveling the underlying mechanisms responsible for regulating
development and disease.

DNA methylation & gene regulation
DNA methylation is an important
regulator of gene expression andchromosome conformation. It occurs
at the cytosine bases of eukaryotic
DNA, which are converted to 5-methylcytosine (5-mC) by DNA methyltransferase enzymes. DNA methylation
occurs almost exclusively in the context
of CpG dinucleotides that are relatively
rare in the mammalian genome andtend to be clustered in CpG islands.
Approximately 60% of gene promoters
are associated with CpG islands and arenormally unmethylated. CpG methylationof gene promoters is usually associatedwith transcriptional silencing, which
can occur through a number of
mechanisms, including the recruitment of methyl binding domain (MBD)
proteins. DNA methylation is involvedin a number of cellular functions, suchas embryonic development, genetic
imprinting, X chromosome inactivationand control of gene expression. Alterations in normal DNA methylation
patterns, either local upregulation
at promoters resulting in silencingof specific cell cycle regulatory ortumor suppressor genes, or a global
decrease in DNA methylation, arehallmarks of various types of cancer.

Methylation & the cancer epigenome
Changes in DNA methylation havebeen well characterized in cancer.
In general, the cancer epigenome ismarked by global DNA hypomethylationand promoter-specific DNA hypermethylation, which often leads tosilencing of tumor suppressor genes.
This link to cancer is supported by thefinding that mutations in DNMT3A are
found in acute myeloid leukemia, andthere are currently two FDA approved
cancer drugs that target DNMTs. The
recently characterized DNA modification 5-hydroxymethylcytosine (5-hmC)
has also been linked to cancer. Both areduction in 5-hmC and in expression of
the TET enzymes that convert 5-mC to
5-hmC have been reported in breast,
liver, lung, prostate and pancreaticcancer.
Tools for DNA methylation analysis
Active Motif offers a number of products
specific for DNA methylation research,
including kits and antibodies to enrichfor DNA fragments that contain 5-mC
and 5-hmC. Our DNA methylationrelated antibodies have been validated
for various applications, including
methylated DNA immunoprecipitation(MeDIP) and immunofluorescence.

www.activemotif.com

EPIGENETICS & CANCER

For complete details on all of our DNA
methylation products, or to download afree copy of our new DNA Methylation& Cancer eBook, please visit us atwww.activemotif.com/dnamt.
The role of non-coding RNAs in cancer
The discovery of non-coding RNAs
(ncRNAs) and their role in gene regulation represents a significant advancein cancer research. These ncRNAs
interact with DNA, RNA or protein
molecules and/or some combination
thereof, acting as essential regulatorsin chromatin organization, and transcriptional and post-transcriptional
regulation. The functional relevanceof ncRNAs is particularly evident for
microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), which havebeen associated with all stages ofcancer including diagnosis, staging,progression, prognosis and responseto treatment. Their misexpression
confers upon a cancer cell the capacity
for tumor initiation, growth andmetastasis, making ncRNAs attractive
therapeutic targets and potentially
useful diagnostic tools.

and the first miRNA mimic entered theclinic for cancer therapy in 2013.
Tools for miRNA analysis
Inherent to the rapid advancements
and general excitement surrounding
miRNA discoveries is the need for
applicable and validated experimental
tools to enable researchers to accurately
study the expression and biological
function of miRNAs. There are a numberof diverse methodologies available for
miRNA detection and target identification,
as well as to determine the functional
effects of a specific miRNA of interest.
These include computational tools,
expression and proteomics assays,
3’UTR reporter assays and chromatin
immunoprecipitation (ChIP) based
techniques. Active Motif offers a wide
selection of tools to enable researchers
to identify mRNA targets and elucidate
Transcription Factors
1. CpG promotor
methylation
DNA

miRNA gene

the functional relevance of a specificmiRNA, including the LightSwitch™
Luciferase Reporter Assay System.
3’UTR reporter assays have become
an important component of thoroughmiRNA target studies because they
provide functional evidence for, andquantitate the effects of, specificmiRNA-3’UTR interactions in a cellbased system. For complete details on
all our miRNA products, or to download
a free copy of our new microRNAs in
Cancer eBook, please visit us at www.
activemotif.com/ncRNAs-and-cancer.
Let us do the work for you Active Motif Epigenetic Services
Our Epigenetic Services team provides
a wide variety of DNA Methylation andGene Regulation services. For more
complete information, or to get a quote,visit www.activemotif.com/services.

2. Loss of normal repressive
epigenetic marks
Genomic loss
Loss of histone acetylation
Aberrant DNA hypermethylation

onco-miRNAs
onco-suppressor
miRNAsMicroRNAs in cancer
MicroRNAs are important epigenetic
regulators of gene expression and
AAA
5 Cap
Pri-mRNA
have been shown to play a role innumerous biological processes, such
3. Drosha downregulation
Cropping
as cellular signaling, cell differentiation,
growth, development and apoptosis.
Mutations and improper regulation ofPre-miRNA
NucleusmiRNAs have been linked to a varietyof physiological disorders, such as
4. XPO5 mutation
XPO5
cancer and heart disease (Figure 1).
Cytoplasm
The recent emergence of miRNAs has
Oncogeneaccumulation
been one of the defining developments
5. DICER1 downregulation
5. TARBP2 mutationin cancer biology. Many studies havedemonstrated how miRNAs impact
7. tumor
6. oncogenic
cancer progression through controlling
suppressor
miRNA
expression of their target mRNAs tomiRNA loss
accumulation
Unwinding offacilitate tumor growth, invasion,
miRNA duplex
angiogenesis and immune response.
The explosion of knowledge since their
Loss of tumor
discovery in 1993 has brought forward
suppressor genes
new diagnostic and therapeutic opportunities. Clinical trials utilizing miRNA Figure 1: DNA methylation and miRNA mysregulation in cancer.
A schematic representation of alterations in DNA methylation and in the miRNA biogenesis pathway that commonly lead to cancer development
profiling for patient prognosis and(adapted from Bertoli G. et al. (2015). Theranostics 5, 1122–43).
clinical response are now underway,sales@activemotif.com

3TOOLS FOR RNA ANALYSISRNA Analysis Techniques to Advance our Understandingof the Transcriptome
Technological advances in the field of RNA analysis have expanded our understandingof the transcriptome beyond messenger RNA (mRNA) to introduce new classes of RNAs
that include short and long non-coding RNA (ncRNA). These non-coding RNAs vary incellular localization and appear to have unique regulatory functions. Combining RNA
analysis with high quality RNA isolation from subcellular fractions, such as with Active
Motif’s RNA Subcellular Isolation Kit, or with next-generation sequencing (NGS) by
utilizing Active Motif’s RNA Sequencing (RNA-Seq) Service, enables you to achieve agreater understanding of RNA function in your biological system.

High quality RNA fractions improvedownstream RNA analysis
Active Motif’s RNA Subcellular Isolation
Kit is designed to efficiently isolate separate nuclear and cytoplasmic fractionsof RNA for downstream analysis. Thismethod can be used to isolate both longand short RNA sequences from cells
or tissue without cross-contaminationor the use of phenolic compounds.
Traditional methods fall short for RNA
analysis because they often isolate total
RNA which consists of a mixture of
intronic RNA originating from immaturetranscripts in the nucleus, mature RNA
from the cytoplasm and a variety of
non-coding RNAs. Such heterogeneity
can bias downstream analysis and

RNA-Seq Service – get the most outof your RNA-Seq experiment
Active Motif provides a comprehensive
RNA-Seq Service that includes
everything from isolation of RNA todata analysis. Combine our worldwide
leading ChIP-Seq Service with RNA-Seq
to gain an even greater understandingof your biological system.
RNA-Seq is the most widely usedtechnique for studying gene expression
changes in response to treatment anddisease, but it also provides muchmore. With sequence data from an
RNA-Seq experiment it is possible to4data interpretation by reducing thesensitivity of detection for low abundance
transcripts and the ability to study
RNA processing dynamics. The RNA
Subcellular Isolation Kit reduces thiscomplexity to improve downstream
analysis and also provides information
regarding RNA localization.
What’s in the box?
Each kit contains enough reagents toperform 15 cytoplasmic and 15 nuclear
RNA isolations. For more information,
visit www.activemotif.com/rna-iso.

RNA Subcellular Isolation Kit• Separates nuclear &cytoplasmic RNA withoutcross-contamination• Works with cell or tissue samples• Spin columns included for
RNA purification• Purified RNA validated for use
in RT-qPCR and RNA-Seq

Product

FormatCatalog No.

RNA Subcellular Isolation Kit

30 rxns

25501

DNase I Treatment Kit

30 rxns

25502

obtain, among other things, information
about splice variants, gene fusions
and SNPs. It is, therefore, important todesign RNA-Seq experiments according
to the goals in mind. Should you use
paired-end or single-end sequencing?
What length of sequencing read isneeded, and how deep should you go?

Is polyA selection or rRNA depletion
appropriate for your experiment?
Active Motif understands and offers
solutions for all the experimental
variables. We will consult with you andprovide specific recommendations on
experimental design that preciselyalign with the goals of your research.

RNA-Seq Service Features• PolyA selection or rRNA reduction• QC performed using Bioanalyzer• Directional library preparation• Data analysis options (TOPHAT,CUFFLINKS, CUFFCOMPARE,
CUFFDIFF)• 50bp or 100bp paired-end reads

www.activemotif.com

TRANSCRIPTION FACTOR SCREENING

NEW

Tools to Study Nrf2 Transcription Factor Activation

Nrf2 activation (OD450 nm)

Nrf2 is an important transcription factor (TF) in the defense against oxidative stress.
Much investment has been made in developing Nrf2 agonists for the treatment ofdiseases such as multiple sclerosis, diabetes and cancer. Like most TFs, Nrf2 activation
is dynamically regulated, involving controlled protein degradation and nuclear
translocation. Active Motif offers a range of products for Nrf2 research, including
TransAM®, a simple, plate-based assay to screen for Nrf2 activation, a chromatin
immunoprecipitation (ChIP) validated Nrf2 antibody and compatible ChIP kit, and aluciferase reporter assay system along with pre-cloned Nrf2-related reporter vectors.

1

0.75

Figure 1: Nrf2 activation is detectedUnstimulated
D,L Sulforaphane Stimulatedusing the TransAM Nrf2 Kit.
Active Motif’s Nuclear Extract Kit wasused to prepare nuclear lysates fromunstimulated HepG2 cells and cells that

0.5

were stimulated for 24 hours with 10 μM
D,L-Sulforaphane. The extracts wereassayed using a range of 0.625 to 10 μg

0.25

per well to show the linearity of theassay. Robust Nrf2 activation is clearly0detectable across all conditions.

0.63

1.25

2.5

5

10

Extract/well (µg)

Screening multiple samples?
Active Motif’s TransAM® Nrf2 Kit includes
a 96-stripwell plate to which an oligomer
corresponding to the antioxidant
response element (ARE) has been
immobilized. Nuclear extracts, preparedusing Active Motif’s Nuclear Extract Kit,
are added to each well. Activated Nrf2
binds the ARE consensus sequence,
while unbound proteins are washedaway. Detection is achieved using an
Nrf2 primary antibody and HRPconjugated secondary antibody alongwith colorimetric detection reagents.
The assay signal is easily measured by
OD 450nm using a microplate reader(Figure 1). In addition to Nrf2, TransAM
is available for over 40 transcription
factor targets. For more information
and a complete list of TransAM products,
go to www.activemotif.com/transam.
Active Motif also offers cell-based
screening assays using oursales@activemotif.com

LightSwitch™ Luciferase Reporter
Assay System that combine optimized
transfection and luciferase assay
reagents along with a large selection of
Nrf2 responsive luciferase reporter
constructs. These include a variety ofsynthetic ARE elements and pre-cloned
reporter vectors containing thepromoters of Nrf2 responsive genes
such as NQO1 and HMOX1.
Interested in monitoring Nrf2
activation across the genome?
ChIP-Seq is now the most widely usedtechnique to study transcription factor
Product

binding. To facilitate your ChIP-Seq
studies of Nrf2, Active Motif provides
a Nrf2 ChIP-Seq validated antibody
and our best-selling ChIP-IT® HighSensitivity Kit (Figure 2). Or let Active
Motif Epigenetic Services do the experiment for you. As the ChIP Experts™,with over 10,000 samples processed
and the highest level of expertise ofany service provider, you can expect
the best results. For more information
about our custom ChIP-Seq services,
visit www.activemotif.com/services.

NQO1

HMOX1

Figure 2: ChIP-Seq was performed using Active Motif’s
Nrf2 antibody and ChIP-IT High Sensitivity Kit.
Select peaks are presented and show Nrf2 binding in the
promoters of known Nrf2 responsive genes.

FormatCatalog No.

®

TransAM Nrf2 Kit

1 x 96 rxns

50296

Nuclear Extract Kit

100 rxns

40010

ARE synthRE

5 μg

32108

NRF2 (NFE2L2) antibody (pAb)

100 μl61599

ChIP-IT High Sensitivity Kit

16 rxns

53040

®

5

CHROMATIN IMMUNOPRECIPITATION

Practical Guide to Chromatin
Immunoprecipitation Techniques

TOPICS
Performing ChIP on epigenetic
modifications
What if I don’t have a sonicator?
What if I have limited sample material?
Analyzing low abundance targets(transcription factors)
Choosing the correct antibody
What if I don’t have a good antibody?
Increasing DNA binding site resolution

How do I normalize for sample bias?
What interactions are occurring at mypromoter of interest?
How can ChIP aid in genome editing?
Can I also interrogate the DNA
methylation status of my ChIP DNA?
Difficult-to-ChIP clinical samples
Analyzing chromatin-associated RNA
NEW: Determine CRISPR/Cas9
specificity using enChIP

Chromatin immunoprecipitation (ChIP) is the most widely utilized
technique for analysis of protein-DNA interactions and histone
modifications. Given the importance of ChIP and ChIP-Seq data
sets for development and disease research, obtaining high quality
data is crucial. However, ChIP can be technically challenging,
and researchers who are not experts in ChIP techniques are best
served using well-validated and reliable kits to perform theseassays. As the ChIP experts®, Active Motif has developed a numberof highly innovative kits and reagents tailored to aid researcherswith their ChIP experiments and to overcome many of the common
challenges researchers encounter when applying this technique.
Performing ChIP on epigenetic
modifications
As a leader in providing innovative
solutions for ChIP, Active Motif has
developed a highly comprehensive
portfolio of ChIP products and services
to make ChIP technologies accessible
to researchers at any level of expertise,
and to also provide cutting-edge
technologies to address many of thecommon limitations experienced by
ChIP users when analyzing chromatinfunction.
Traditional ChIP and ChIP-Seq are extremely informative methods for identification and profiling of chromatininteractions with ubiquitous features,for example chromatin modifying
proteins (e.g. p300, HDACs), histone
modifications (e.g. H3K4me3, H3K27me3,
H3K27ac) and transcriptional machinery(e.g. RNA pol II). When conducting
ChIP analysis, the most important
aspects of your ChIP protocol are that it
be reproducible, sensitive and efficient.
For performing ChIP on relatively
abundant proteins such as histones,
Active Motif’s ChIP-IT® Express kits
provide the ideal solution. ChIP-IT
Express kits use magnetic beads tostreamline the ChIP protocol, allowing

6

the ChIP procedure to be completed
in a single day. The reduced hands-ontime and use of ChIP-grade magnetic
particles minimize DNA loss andsample variation, enabling ChIP to be
performed on as few as 100,000 cells.
With over 500 citations in peer reviewed
publications, this is the method of
choice for routine ChIP (Figure 1).
What if I don’t have a sonicator?
The kit comes in two formats, the original ChIP-IT Express sonication protocol
and ChIP-IT® Express Enzymatic, the first
kit that enables shearing of chromatin
by enzymatic digestion rather thansonication for users who do not routinely
perform ChIP and either do not own asonicator or are not proficient in its use.
To learn more about ChIP-IT Express,
visit www.activemotif.com/chipit.
What if I have limited starting material?
Standard ChIP-Seq protocols requireat least 10 million cells to perform
ChIP-Seq, a number that is unattainable
for many cell types. As a result, oneof the major obstacles preventing
researchers from generating quality
ChIP-Seq data is the limited cell numbers available for their experiments.
Active Motif’s Epigenetic Services groupwww.activemotif.com

# of Publications

Sigma
Imprint®

Diagenode
LowCell#

Invitrogen
MAGnify™

Millipore
Magna ChIP™

Active Motif
ChIP-IT® Express

050

100 150 200 250 300 350 400

CHROMATIN IMMUNOPRECIPITATION

Figure 1: ChIP-IT Express is the most highly publishedcommercial ChIP kit on the market. Comparison of
ChIP-IT Express citations vs. competitor kits.

has made significant improvements to
our ChIP-Seq protocols and pipeline
and, as a result, reduced our starting
material requirements by 1,000 fold.
Our Low Cell Number ChIP-Seq Service
enables generation of genome-widebinding profiles from as few as 10K cells.
For more information, please visit us atwww.activemotif.com/lowcellcs.
Analyzing low abundance targets(transcription factors)
Performing ChIP-Seq on transcription
factors is often problematic due to therelative low abundance of these proteinsand quality of available antibodies. To
address this, scientists at Active Motifhave developed the ChIP-IT® HighSensitivity Kit, the most sensitive ChIP
kit available on the market. The kit utilizes specially formulated reagents for
preparation of high-quality chromatinfrom minimal amounts of cells or tissue (1000 cells for abundant targets
and 50,000 cells for low abundance
transcription factors per reaction;
the chromatin preparation module isalso available separately as the HighSensitivity Chromatin Preparationkit). During the immunoprecipitation(IP) reaction, low-background protein
G agarose beads and an antibody
blocker reduce non-specific binding.
Specialized ChIP buffers enhanceenrichment and reduce non-specificsales@activemotif.com

DNA interactions. ChIP filtration gravity
flow columns are used for faster,
simpler and more consistent capture
and wash steps. The result is a kit thatis capable of delivering both higher
signals and reduced background levels
when compared to other commerciallyavailable ChIP kits. To learn more
about ChIP-IT High Sensitivity, visit
www.activemotif.com/chipiths.
Choosing the correct antibody
Another challenge for epigenetics
research is the lack of antibodies thathave been validated for use in ChIP and
ChIP-Seq. The problem is compoundedby antibody suppliers who do not
manufacture or test the antibodies
they sell, and who sell them to one
another and then to researchers. At
Active Motif, we manufacture andrigorously test our antibodies in-houseto ensure their quality and performance.
Our ChIP antibodies are validated
according to the guidelines of the
ENCODE* Consortium and run throughthe same internal validation programused by our ChIP-Seq Services group.
For a complete, up-to-date list ofavailable antibodies, please visit us atwww.activemotif.com/chipabs.
*For ENCODE Consortium guidelines for antibody validation,please refer to Landt S.G. et al. (2012) Genome Res. 22,
1813–1831.

What if I don’t have a good antibody?
ChIP analysis is often hindered by thelack of available antibodies capable ofrecognizing fixed protein targets, orthe inability of antibodies to distinguishbetween protein isoforms. Active Motif
has developed the Tag-ChIP-IT® Kit toenable ChIP without the use of a targetspecific antibody. Tag-ChIP-IT utilizes
a unique AM-tag that, unlike traditional
FLAG, GFP or HA tags, is specifically
optimized for ChIP. Furthermore, we havedesigned a high specificity antibody tothe tag to optimize pull-down efficiency
during immunoprecipitation (Figure 2).
The AM-tag is engineered to have minimal cross-reactivity with mammaliansamples. Additionally, in contrast tolarger GFP and FLAG tags, the smaller
footprint of the AM-tag reduces obstruction of the DNA binding domain. The
AM-tag design also maximizes exposure
during the IP reaction to increase theenrichment efficiency of low abundance
transcription factors for more reliable
and consistent ChIP results. To learn
more, visit www.activemotif.com/tagchip.
Increasing DNA binding site resolution
For researchers working with transcription factors, another common
issue encountered with ChIP, aside
from low abundance of protein, isthe poor resolution of binding sites.
Traditional ChIP-Seq methods only

Tag-ChIP-IT Identifies Estrogen Receptor (ER) Binding Motifs

ER Motif Identified

SampleAM-tag ER#1

AM-tag ER#2

Figure 2: ER cDNA was cloned into pAM_1C Empty Vector and transiently transfected into cells. Cells were induced with estradiol
and chromatin was harvested and Tag-ChIP performed using the Tag-ChIP-IT Kit. Following cross-link reversal, enriched DNA was
submitted for next-generation sequencing. Data was compared to published anti-ER antibody ChIP-Seq results using the same cellline and induction conditions. ChIP-Seq data shows the same ER peak profile with the AM-tag ChIP as endogenous ER. Detectedbinding sites were further evaluated for binding motifs. Results show the ER motif was identified in both Tag-ChIP-IT samples.

7

CHROMATIN IMMUNOPRECIPITATION

applied across samples and antibodies
to eliminate bias and reveal any realor obscured biological differences.
ChIP normalization can easily be
implemented by integrating our Spike-in
reagents into your existing ChIP protocol.
For more complete information, go towww.activemotif.com/spikein. To learn
about custom Spike-in Services, visit
www.activemotif.com/services-normalize.

Active Motif’s
ChIP-exo
ChIP-seq
ChIP-chip

-300

0

300

Distance from motif (bp)
Figure 3: ChIP-exo achieves significantly higher resolutionof DNA binding sites than traditional ChIP methods.

resolve DNA binding sites within ≥200
bp when the average DNA binding site
length of individual transcription factors
tends to be in the region of 6-10 bp.
To address this, Active Motif offers the
ChIP-exonuclease (ChIP-exo) Kit containing an optimized and streamlined
protocol for high-resolution genomewide mapping of transcription factor
binding sites. ChIP-exo is a modified
ChIP-Seq approach that enables
researchers to identify protein-DNA
binding sites at a resolution of 20-95
base pairs, refining the ability to identify
DNA motifs more precisely and distill
genome-wide protein binding profiles
for studies of mutational or SNP
effects on transcription factor bindingsites (Figure 3). For more information,
go to www.activemotif.com/chipexo.
How do I normalize for sample bias?
ChIP-Seq is a multi-step process inwhich disparities caused by sample
loss during immunoprecipitation andlibrary preparation, uneven sequencing
read depth or technical variation canlead to results that are difficult tointerpret. To overcome this challenge,
Active Motif has developed a ChIP
Normalization Spike-in strategy fornormalization of ChIP qPCR and
ChIP-Seq data to reduce the effects
of technical variation and sample
processing bias. This strategy can be

8What interactions are occurring at
my promoter of interest?
When performing ChIP experiments, it
is often useful to analyze co-occupancy
of proteins or epigenetic modifications
at a given genomic locus. Active Motif’s
Re-ChIP-IT® Kit is a Sequential ChIP
(Seq-ChIP, Re-ChIP) procedure inwhich ChIP samples are subjected tosequential rounds of immunoprecipitations using two different antibodies
to determine whether proteins orepigenetic modifications co-occupy
the same genomic region. The Active
Motif Re-ChIP-IT Kit utilizes the samemagnetic bead-based protocol usedin our ChIP-IT Express Kits to provide
a streamlined Re-ChIP protocol forsequential ChIP analysis. Unlike other
Seq-ChIP methods, the Re-ChIP-IT
method eliminates the need for direct

Experimental
Chromatin

Spike-in
Chromatin
Spike-in
Antibody

Chromatin Immunoprecipitation

Sequencing

Map to experimental
genomeNormalize sample
tag counts by same ratio

Active Motif’s recent addition to it’ssuite of custom services is RIME (Rapid
Immunoprecipitation Mass Spectrometry of Endogenous Proteins). RIME
resolves the complex process of gene
regulation by enabling the capture
and identification of interactomes, orthe associated protein networks, of an
endogenous protein of interest. Gene
regulation research is often oversimplified by solely focusing on one
regulatory factor. In reality, differential gene expression is the result of
the highly orchestrated and complex
interactions of chromatin modifiers,
epigenetic modifications, coactivatorsand repressors, and other regulatory
elements with chromatin. The RIME
service includes immunoprecipitationof a target protein from cross-linked
cell extracts followed by ChIP-Seq,
mass spectrometry and data analysis.
To learn more about our RIME Services,
visit www.activemotif.com/rime.
How can ChIP aid in genome editing?

ChIP-Seq Normalization Workflow

Antibody ofinterest

antibody conjugates or an immunoselection matrix and includes specialized
buffers that prevent carryover ofprimary-to-secondary immunoprecipitation reactions. To learn more, visit
www.activemotif.com/rechip.

Map to Drosophila
genomeNormalize Drosophila
tag counts across samples

CRISPR/Cas9 genome editing has
gained prominence in genetic researchand has expanded its capabilities in somany ways, providing a simple method
to potentially genetically modify
plants, eradicate viruses, and identify
cancer genes. However, there areunintended consequences of thistechnology, mainly issues with Cas9
nuclease specificity in creating doublestranded breaks leading to off-target
effects. To enable you to assess Cas9
specificity, Active Motif’s enChIP Kit(Engineered DNA-binding moleculemediated Chromatin Immunoprecipitation) offers a modified ChIP
assay that utilizes the CRISPR/Cas9
system to target a specific DNA locus
for immunoprecipitation. Using the
enChIP Kit for your genome editingwww.activemotif.com

CHROMATIN IMMUNOPRECIPITATIONRIME Service Workflow for Identification of Endogenous Interactomes

Cross-link cells

Sonicate

Isolate nuclei

Identify bindingsites by ChIP-Seq
Immunoprecipitate target
Identify interacting
proteins by mass
spectrometry

experiments enables you to biologicallyvalidate each target sequence for
specificity. Sequences demonstrating
off-target effects can be excluded,
saving valuable time and resources.
To learn more, go to page 10 or visit us
at www.activemotif.com/enchip.
Can I also interrogate the DNA
methylation status of my ChIP DNA?
For those interested in DNA methylation profiles of their genomic region ofinterest, Active Motif’s ChIP-BisulfiteSequencing (ChIP-Bis-Seq) Kit offersa method to directly interrogate both
chromatin and DNA methylation status
within the same sample. ChIP-Bis-Seq
combines a ChIP procedure for targetspecific enrichment of protein-boundor modified chromatin with bisulfite
conversion and sequencing (Bis-Seq)
to allow single nucleotide resolution
DNA methylation profiles of ChIPenriched chromatin. This approachis ideal for genome-wide studies of
the interactions between DNA methylation and chromatin features, suchas in assessing allele-specific DNA
methylation variances (e.g. imprinting,X-inactivation), or in evaluating global
gene regulatory effects. To learn more,
visit www.activemotif.com/chip-bis-seq.
Difficult-to-ChIP clinical samples
In disease research, when attempting

sales@activemotif.com

to harvest valuable biological informationfrom clinical samples, many of the common clinical sample types are primarytissues that present a challenge either
because of the difficulty in extracting
high-quality chromatin from difficultto-lyse cells, very limited amounts ofsample, or highly degraded material.
Active Motif provides the ChIP-IT® FFPE
and ChIP-IT® PBMC assays to finally enable researchers to perform ChIP analysis of primary tissue samples. These
ChIP assays are the only commerciallyavailable assays specifically developed
and optimized to extract high-quality

chromatin from primary cells for ChIP
analysis and next-generation sequencing. For more information on the
ChIP-IT FFPE and PBMC kits, please
visit us at www.activemotif.com/ffpe
or www.activemotif.com/pbmc.
Analyzing chromatin-associated RNA
For researchers interested in the roleof RNA in regulation of the genome,
Active Motif has adapted the magnetic
bead-based method used in our ChIP-IT
Express Kits for the development of
the first-of-its-kind kit for RNA-ChIP.
The RNA ChIP-IT® Kit is specificallydesigned to study RNA-protein interactions in a chromatin context. RNA
ChIP-IT’s innovative fixation method issuperior to existing RIP technologies
that use native nuclear extracts because
fixation preserves chromatin interactions, taking into account the dynamic
chromatin environment of the RNAprotein complexes of interest. For more,
visit www.activemotif.com/rnachip.

For a complete list of available
ChIP products & services,
please visit us atwww.activemotif.com/chip

Product

FormatCatalog No.

ChIP-IT Express Kit

25 rxns

53008

ChIP-IT Express Enzymatic Kit

25 rxns

53009

ChIP-IT High Sensitivity Kit

16 rxns

53040

High Sensitivity Chromatin Preparation Kit

16 rxns

53046®

Tag-ChIP-IT Kit

16 rxns

53022

ChIP-exo Kit

12 rxns53043

Spike-in Antibody

50 µg

61686

Spike-in Chromatin

10 µg

53083

Re-ChIP-IT Kit

25 rxns

53016

ChIP-Bis-Seq Kit

10 libraries

53048

ChIP-IT FFPE Kit

16 rxns

53045

ChIP-IT PBMC Kit

16 rxns

53042

RNA ChIP-IT Kit

25 rxns

53024

®

®

®

®

®
®

®

9

ChIP & CRISPR/Cas9 GENE EDITINGNEW

Determine CRISPR/Cas9 Specificity Using enChIP
enChIP* is a modified chromatin immunoprecipitation (ChIP) assay that utilizes the
CRISPR/Cas9 system to target a specific DNA locus for immunoprecipitation.
Using the enChIP Kit enables you to biologically validate eachtarget sequence for specificity. Sequences demonstrating
off-target effects can be excluded from genome editing
experiments, thereby saving valuable time and resources.
*Patent number: WO2014/125668

How does enChIP work?
Active Motif’s enChIP Kit (Engineered
DNA-binding molecule-mediatedChromatin Immunoprecipitation)utilizes the CRISPR/Cas9 system todirect a guide RNA (gRNA) to a specificgenomic locus for immunoprecipitation.
The gRNA is co-expressed with an
enzymatically inactive form of the
Streptococcus pyogenes Cas9 endonuclease (dCas9). The dCas9 protein
contains Active Motif’s unique AM-tag
sequence that was designed specifically for high sensitivity enrichment
in ChIP. Following transfection of thegRNA and AM-tagged dCas9, cells areformaldehyde fixed, lysed, and thechromatin is fragmented by sonication.
A monoclonal antibody directed against
the AM-tag is used to enrich for genomic

enChIP Kit Highlights• Identify off-target binding events

enChIP Kit Validates
enChIPSpecificity
Kit Validates gRNA Specificity

Figure 1: Two gRNAs were designed targeting
different 20 nt sequences within a 500bp

Target Site

CTCF

region of a CTCF binding site on chromosome
19. Each gRNA sequence was cloned into
the pAM_dCas9 vector and transfected into

ChIP-Seq

HEK293T cells. A ‘No gRNA’ negative control
was also performed. Chromatin was preparedgRNA2

and immunoprecipitated according to the
enChIP Kit. Enriched DNA was analyzed by
NGS and background was subtracted using

gRNA1

the ‘No gRNA’ control. Data was compared to
ChIP-Seq data for CTCF in the same cell line.
Results show gRNA1 had a strong peak andNo gRNA

specific binding at the target location. Data for
gRNA2 revealed a large number of off-target
binding events outside of the target location.
This confirms that enChIP provides biologicalvalidation for CRISPR/Cas9 specificity.

sequences bound by the gRNA/dCas9
complex. Following immunoprecipitation, purified DNA can be analyzed
by qPCR or NGS to evaluate binding
specificity or chromosomal looping.
For more information, please visit us
at www.activemotif.com/enchip.

What’s in the box?
The enChIP Kit contains chromatin
preparation reagents, enChIP buffers,
AM-tag monoclonal antibody, protein G
magnetic beads and DNA purification
reagents for 16 enChIP reactions. The
gRNA and dCas9 expression vectors
are sold separately.

• Evaluate chromosomal looping• Versatility to use your own gRNA
constructs or clone into one of
Active Motif’s vectors• Utilizes AM-tag specificallydesigned for use in ChIP
• High sensitivity assay detects
sequences present at only 1 or
2 genomic locations

10

Product

FormatCatalog No.

enChIP Kit

16 rxns53125

pAM_gRNA Vector

10 µg

53121

pAM_dCas9 Vector

10 µg

53122

pAM_gRNA_CTCF Vector (Positive control)

10 µg

53123

pAM_dCas9_CTCF Vector (Positive control)

10 µg

53124

FuGENE HD Transfection Reagent

0.2 ml32042®

www.activemotif.com

GLOBAL DNA METHYLATION

Quantify Global Changes in 5-mC & 5-hmC in
Normal and Diseased Samples
The cancer epigenome is marked by alterations in a myriad of epigenetic features,
including DNA methylation, nucleosome positioning and histone post-translational
modifications. These changes influence gene regulation and can serve as signaturesof cancer. To better understand how changes in DNA modifications influence normal
and diseased states, Active Motif offers simple, easy-to-use assays to perform
comparative studies of global changes in 5-methylcytosine (5-mC) and
5-hydroxymethylcytosine (5-hmC).

enzymatically digested to create LINE-1
fragments. The DNA is then hybridized
with a LINE-1 consensus probe.
Following capture onto a 96-well plate,a 5-mC antibody and colorimetricdetection reagents are used to generate
a signal that is easily measured with amicroplate reader. The assay includes
DNA standards for 5-mC quantification and can detect as little as 0.5%
methylcytosine (Figure 1).
Global DNA Methylation – LINE-1

COMING SOON:
Global LINE-1 Kit
for MURINE samples

sales@activemotif.com

0.9

Global 5-hmC Quantification

0.8
0.7

2

% 5-hmC

0.6

1.5
1

Global 5-hmC ELISA
Mass Spectrometry

0.5
0.4
0.3

0.5
0

0.2
0.1
Blank 0% 1.3% 2.6% 3.9% 6.6% 9.9% 13.1%

HeLa HeLa+ Aza% 5-mC of total cytosine content (100 ng/well DNA)

Quantify changes in global 5-mC levels
Active Motif’s simple, plate-based
Global DNA Methylation – LINE-1 Kit
uses a consensus sequence within thehuman repetitive Long Interspersed
Nucleotide Element 1 (LINE-1) as asurrogate readout for global 5-mC
levels. First, genomic DNA is

Quantify changes in global 5-hmC levels
Using Active Motif’s Global 5-hmC
Quantification Kit, hydroxymethylcytosine
levels can be quantified from as little
as 20-50 ng of genomic DNA. Thiseasy-to-use plate-based assay offers
higher throughput quantification using
less input material than mass
spectrometry and can detect as little
as 0.02% 5-hmC with the included
DNA standards (Figure 2).

2.5

OD 450nm

Global DNA methylation & disease
In mammals, DNA methylation ischaracterized by the covalent additionof a methyl group within the cytosineof a CpG dinucleotide. These CpG
sites are often found within repetitive
regions and are normally methylated
to repress transcription. In cancer,
hypomethylation of these repetitiveelements is observed. Likewise, the DNA
methylation variant 5-hmC is known toplay a role in transcriptional regulationand embryonic development and may
also serve as a prognostic indicator incertain cancers and neurodegenerative
disorders. Hydroxymethylcytosine
results from the oxidation of 5-mC
by the Ten Eleven Translocation (TET)
enzymes. Because of their importancein biological processes and disease,methods to accurately quantify global
5-mC and 5-hmC are highly warranted.

0mouse mouse mouse mouse mousebrain heart kidney liverlunghuman human humanbrain kidney liver

Figure 1: The Global DNA Methylation – LINE-1 Kit was

Figure 2: The Global 5-hmC Quantification Kit was used

used to compare the methylcytosine levels between two

to determine the % 5-hmC in genomic DNA isolated

genomic DNA samples. Assay results show a decrease

from mouse and human tissues. Genomic DNA wasin 5-mC levels resulting from treatment of HeLa cells

tested at 20 ng/well for human brain and 50 ng/well forwith 5-azacytidine (Aza), a DNA methyltransferase inhibitor.

all other DNA samples. The % 5-hmC was determined

The % 5-mC is calculated using the included DNA standards.

using the included DNA standards. Results were

Methylcytosine levels of the DNA standards are displayedcompared to mass spectrometry data obtained from 500 ng

as a percentage of total cytosine content.

of the same DNA samples. Results show the Global
5-hmC Kit provides equivalent quantification using only afraction of the starting material.

Product

FormatCatalog No.

Global DNA Methylation – LINE-1 Kit

1 x 96 rxns

55017

Global 5-hmC Quantification Kit

1 x 96 rxns

55018

11

miRNA TARGET IDENTIFICATIONA Genome-wide 3’UTR Collection for High-throughput
Functional Screening of miRNA Targets
To enable researchers to study microRNAs (miRNAs) using 3’UTR-reporter assays
on a high-throughput scale, we have created a genome-wide collection of 12,000
human 3’UTR luciferase reporters in the highly-optimized LightSwitch™ Luciferase
Assay System. The assay system is ideal for performing miRNA target validation andassessing the functional impact of miRNA-3’UTR interactions. Combined with ourlarge collections of miRNA Mimics & Inhibitors, you have everything needed to study
miRNA-3’UTR interactions, validate miRNA targets, and measure RNA stability andthe functional impact of miRNAs on a gene-by-gene basis.

miRNA mimicor
non-targeting control

co-transfect

3’UTR
Reporter

RISC
complex

HybridLUC-3UTR
Transcript

Luciferase-based screening to
identify miR-122 targets
The LightSwitch system represents
a sensitive, specific and economicalmeans to probe miRNA-UTR interactions and functionally validate
miRNA targets. To demonstrate, we
performed a high-throughput screen
to identify new targets of miR-122.
Target predictions were obtained
from commonly used sources such as
Miranda and Target Scan. Individual
3’UTR luciferase constructs (Catalog
No. 32011) were employed in co-transfection experiments with either an
1.5

miR-122 mimic (Catalog No. MIM0136)
or a non-targeting control (Catalog
No. MIM9001). Luminescence for
58/142 (40.8%) of the predicted
targets was significantly different in
the mimic co-transfection comparedto the non-targeting control (p<0.05)(Figure 2). Thus, not only did thisscreen identify numerous novel targetsof miR-122, but we also found several
targets with more potent interactions
with miR-122 than previously observed.
These studies exemplify how ourgenome-wide library of human 3’UTRluciferase reporter constructs enables
researchers to screen thousands ofpotential miRNA targets and understand the roles of miRNAs in a single
experiment. To learn more, please visit
us at www.activemotif.com/ls-3utr.

Predicted 3´UTR targets

1.0

miRNA luciferase activity

luciferase 3’UTR
promoterthroughput scale, we have created agenome-wide collection of 12,000
human 3’UTR luciferase reporters in
the highly optimized LightSwitch
Luciferase Assay System (Figure 1).

log2 ratio of targeting/non-targeting

LightSwitch™ 3’UTR reportercollection and assay system
Identification and validation of miRNAtargets can be a complicated task.
There are several alternatives available,
starting with numerous target prediction
methods and continuing with different
biological procedures that encompass
reporter-based screenings, shotgun
proteomics and Ago2-based immunoprecipitation methods. 3’UTR-reporter
assays have become an importantcomponent of thorough miRNA target
studies because they provide functional evidence for, and quantitate
the effects of, specific miRNA-3’UTR
interactions in a cell-based system.
Thus, to enable researchers to leverage
3’UTR-reporter assays on a high-

0.5
0.0
-0.5
-1.0
-1.5

Positive control 2
Positive control 1

-2.0
-2.5

Figure 1: Experimental design for an miRNA target

Figure 2: A functional screen of 142 human 3’UTRs with predicted miR-122 sites in HT-1080 cells. 44 of the targets decreased

functional screen using the LightSwitch System.

significantly by t-test (p<0.05) and 24 were significantly repressed >2-fold.

12

www.activemotif.com

ANTIBODIES FOR CANCER RESEARCH

Validated Antibodies Reveal Histone-mediated Effects
on miRNA in Non-small Cell Lung Cancer
Research has shown that dysregulation of microRNA (miRNA) function is associatedwith many cancers. Furthermore, epigenetic silencing via DNA methylation can alter
miRNA expression. In a recent study led by the laboratory of Dr. Nobuya Ohishi at the
University of Tokyo Hospital, researchers looked at miR-139, a known tumor suppressor
in several cancers, to study its function in non-small cell lung cancer (NSCLC). Using
chromatin immunoprecipitation (ChIP) and Active Motif antibodies to trimethylated
marks on Histone H3, they suggest that H3K27me3-mediated silencing of miR-139
may represent a promising biomarker of lung cancer metastasis.

Histone methylation-mediated silencingof miR-139 enhances invasion of NSCLC
MiR-139-5p (denoted thereafter as
miR-139) has recently been reported
to function as a tumor suppressor inseveral types of human cancers, but its
function in NSCLC and the mechanismof its suppression have not been studied
in detail. In a recent publication, research
conducted in the laboratory of Dr.
Nobuya Ohishi reveals that miR-139 is
epigenetically silenced by histone H3lysine 27 trimethylation (H3K27me3) of
its host gene PDE2A, and that transcriptional regulation is independentof promoter DNA methylation.

Figure 1: Structure of the PDE2A genomic locus. The
locations of miR-139 and the ChIP primers are shown.

For more detailed information, go to
www.ncbi.nlm.nih.gov/pubmed/26256448.
To support their findings, researchers
first confirmed the suppression of thehost gene PDE2A, and the associationof expression levels with miR-139, by
measuring their expression acrossseveral lung cancer cell lines, as well
as NSCLC and adjacent normal lungtissue, using RT-PCR.
With coordinated expression between
miR-139 and PDE2A established, they
then turned to chromatin immunoprecipitation to look at histone methylation
around the promotor of the PDE2A
short transcript (Figure 1) to determine
if H3K27me3 was silencing PDE2A.
Bisulfite sequencing was also used tosales@activemotif.com

Figure 2: ChIP analysis of H3K4me3, H3K27me3, andH3K9me3 in five lung cancer cell lines and normal humanbronchial epithelial cells. IP/Input values were normalized
to a control locus (ACTB promoter).

rule out any dependencies between
histone and DNA methylation.
To form a complete picture of transcriptional regulation, ChIP was performed on histone H3 trimethylated atlysine 4 (H3K4me3), a mark of active
transcription, and lysine 9 (H3K9me3)
which, along with H3K27me3, represent
transcriptional repressors. Aided by
Active Motif’s ChIP validated antibodies
to all 3 marks, ChIP analysis showedno real change for H3K9me3 acrossall cell lines and a positive correlation
between H3K4me3 and PDE2A. However, H3K27me3 had a strong negative
correlation, more so with NCI-H2170
cells with the lowest expression
levels of PDE2A (Figure 2). To further
demonstrate this correlation, siRNAinhibition of EZH2 H3K27me3 methyltransferase led to an increase in
PDE2A in cancer cells, leaving the
researchers to conclude, at least inpart, that H3K27me3 represents apromising biomarker of lung cancer
metastasis.
For a complete up-to-date list ofavailable antibodies, please visit us atwww.activemotif.com/abs.
This article is a summary of the research performed by
Watanabe et al. at the University of Tokyo Hospital, Hongo,
Bunkyo-ku, Tokyo, Japan, published in Cancer Medicine(2015) 4,1573–1582.
This is an open access article under the terms of the Creative
Commons Attribution License.

Product

FormatCatalog No.

Histone H3K4me3 antibody (pAb)

100 µg39915

Histone H3K9me3 antibody (pAb)

100 µl39161

Histone H3K27me3 antibody (pAb)

100 µg39155

13

RECOMBINANT PROTEINSHigh Quality Bromodomain Proteins for Developing
Robust Assays for Epigenetic Drug Discovery
Bromodomain proteins play an integral role in the regulation of transcription andchromatin remodeling. Because BET (Bromodomain and Extraterminal domain) proteins
have been shown to also regulate transcription of certain oncogenes, they are promisingtherapeutic targets for cancer. As a result, there has been an increase in focus on
bromodomain proteins in drug discovery research that has led to the discovery of aclass of small molecule BET inhibitors. Early successes of BRD4 inhibition has peakedinterest in the therapeutic potential of other BET inhibitors. To support research efforts,
Active Motif offers a comprehensive collection of recombinant bromodomain proteins.

BET family members are promisingtherapeutic targets
The BET family of bromodomain proteinshas been shown to play a key role in thedevelopment of several kinds of cancer.
Drug discovery efforts have led to theidentification of multiple small molecule
inhibitors that target BET proteins, andseveral of these inhibitors currently haveadvanced to clinical trials.
BETs are a subclass of proteins thatare characterized by two N-terminal
bromodomains and one ET (Extra
Terminal) domain. The ET domain functions
as a protein binding motif and exerts
atypical serine-kinase activity, while thetwo N-terminal bromodomains, also known
as BD1 and BD2, recognize acetylated
lysines on histones and function to
recruit other BET proteins to chromatin.
Because the recruitment and targetingof serine-kinase and other activities by
BETs to chromatin involve the concerted
interplay of several BET domains, it is
crucial that we understand the effects ofexisting inhibitors on individual domainfunctions.
The quest for inhibitors of BET function
One of the most widely studied BET
inhibitors is JQ1, a small molecule inhibitorof BRD4 with nanomolar affinity thatshows promise across many BRD4
dependent cell lines. As an effective
anti-proliferative agent, JQ1 acts as acompetitive binder, displacing BRD4 from14

chromatin resulting in MYC downregulation.
JQ1 has also been shown to target BRD2
in hematologic malignancies and inhibit
expression of BRD3 and BRD4 in Tamoxifen
resistant breast cancer cells, giving it theability to perform as a pan-BET inhibitor.
I-BET151 and I-BET762 are similar to
JQ1 in that they possess nanomolar
affinity and show strong anti-proliferative
effects. I-BET151 also functions as apan-BET inhibitor showing anti-tumor
activity in multiple myeloma, malignant
brain tumor and other murine models.
I-BET762 is considered a more specificinhibitor and first showed promise in
NUT midline carcinoma (NMC). Phase I
clinical trials for I-BET762 are ongoingfor NMC as well as refractory hematologic
malignancies.

Active Motif Bromodomain Proteins• Over 40 reader domains available• Specifically designed for
epigenetic drug discovery• Custom & bulk orders available• Lot specific data, including:- Activity by HTRF
- Purity by SDS-PAGE
Due to the preliminary success of these
therapeutics in the treatment of cancer,
there is much interest in manipulating
specific domain interactions as a novel
approach for identifying bromodomain
targeted drug therapies. However,there is still much unknown regarding
BET proteins and the individual BDs.

Bromodomains
purity by SDS-PAGE

BRD2 (71-194)
kDa

55
40
35
25

BRD3 (306 - 416)
kDa

55
40
35
25

15

BRD4 (44 -168)
kDa

55
40
35
25
15

15

Figure 1: Purified recombinant proteins were run on an SDS-PAGE gel and stained with Coomassie blue. Coomassie
stains reveal that the recovered proteins are intact with little or no detectable degradation. All proteins were
determined to have ≥ 98% purity.

www.activemotif.com

RECOMBINANT PROTEINS

Phylogenetic Tree Diagram of the Human Bromodomain Family

BRD4 (44-168) Titration

15K

250

PF

D9

BRD7

BRD4 (333-460) Titration

15K

BR

BR

300

3

KIA

D3

BR

BR

A12

DT

200

BRD4

150

Bromodomain conc. (nM)

BRDT

100

BA
Z1A

50

P

0

BB
CRE

0

BR
D2
BR
D3

D1

5K

EP300

BRPF1

BR

HTRF Signal

10K

40

BRD2

ATAD2
BR

D8

BRWD1

5K

IP

50

100

150

200

250

BA

300

1

F
TA

B
KC

PR

B
Z1

B

0
0

P1

3

D
RW

PH

BRD4

BRDT

HTRF Signal

10K

TAF1L
TAF1
TAF1
L

Bromodomain conc. (nM)

BAZ2B

PCAF

5

Z

2

ZM

GCN

L
FA

BA

CR

11
YND

Z2A

(333-460) bromodomains (bottom panel) tested

CE

Figure 2: BRD4 (44-168) (top panel), and BRD4
by HTRF. Assay conditions for bromodomain (BRD)
activity were as follows: 3.3 uM histone peptide
H4K5/8/12/16(tetra-acetyl) was incubated with

MLL
0

)

SP14

PB

)

(2

PB1 (5)

)

1

AR

(4

PB

CA
2

1

SM

3)

1)

A4
RC

A

SM

1(

1(

PB

sales@activemotif.com

L

PB

Active Motif has responded to this needby providing the most comprehensive andvalidated collection of reader domain proteins for use in the development of robust
assays for bromodomain drug discovery.
The most comprehensive reader domaincollection available for drug discovery
As a global leader in providing new andinnovative products and services to enable
epigenetics research, Active Motif offers acomprehensive portfolio of recombinant
epigenetic enzymes and reader domains
for drug discovery research (Figure 3).
Our recombinant proteins are manufactured to meet the highest standards ofpurity (Figure 1) and activity required forconventional drug discovery assays, suchas HTRF (Figure 2), mass spectrometry,
AlphaLISA and Alphascreen. These assays
are routinely incorporated as part of ourquality control process to ensure that

H1

0L

14

SP

BRW
D3
PH
IP

AS

PB1 (6

28

66

IM

SP100

Research and inhibitor screening has
markedly increased in recent years
suggesting a need for validated products
to support these efforts.

1

TIF

TR

used to detect the reaction products.

BRWD1

SP110

at room temperature. Anti-FLAG antibody wasIM

50mM HEPES-NaOH pH 7.0, 0.1% BSA for 1 hour

TRIM33TR

the protein indicated in reaction buffer containing

Figure 3: Active Motif provides a comprehensive collection of high quality reader domain proteins for use in assay
development in early drug discovery. Bromodomain proteins, designated with purple dots, represent currently available
bromodomains in our collection. In addition, as part of our integrative solutions for epigenetic drug discovery,
Active Motif also offers custom and bulk orders, as well as antibodies and inhibitors to these proteins.

our recombinant proteins and enzymes
demonstrate the nM range activities
needed for use in identification of potent
inhibitors.
At Active Motif, we have optimized our
protein production and testing to facilitate
seamless integration of our recombinant
proteins into drug discovery pipelines. We
strive to instill confidence in our proteinsby providing comprehensive application
data that is relevant to drug discovery

platforms and unparalleled customer
support for our drug discovery partners.
For more information and an up-to-date
list of available proteins, please visit
www.activemotif.com/proteins.

To inquire about
Custom and Bulk Orders
CALL: 877-222-9543

15

PRSRT STD
US POSTAGEPAID
PERMIT #94
SAN DIEGO, CA

NORTH AMERICAJAPAN

EUROPE

CHINA

Toll Free: 877 222 9543
Direct: 760 431 1263
Fax: 760 431 1351
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japantech@activemotif.com

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Enabling Epigenetics Research