Ribbon diagram of the nucleosome core particle structure (H2A.Z nucleosome, pdb entry 1F66) viewed down the superhelical axis (left) androtated 90° (right). Original ﬁgure prepared by Dr. Karolin Luger, Department of Biochemistry and Molecular Biology, Colorado State Univ.

IN THIS ISSUE

Antibodies, Kits and Services for Epigenetic Research
2 NEW: ChIP-IT® High Sensitivity Kit for
Difﬁcult-to-ChIP Transcription Factors

9 Recombinant Proteins to Analyze Histone
Post-translational Modiﬁcations

3 NEW: qPCR Analysis Strategy to Normalize
ChIP Data for Easier Interpretation

9 Recombinant Histone Proteins to Analyze
Site-speciﬁc Modiﬁcations

4 NEW: Enhanced Gene Regulation
Analysis with NOMe-Seq

9 NEW: Recombinant Bromodomain
Proteins to Study Bromodomain Function

5 NEW: MethylCollector™ Ultra-Seq is
Now Available as a Service

10 Reagents for High-quality
Fluorescence Microscopy

5 NEW: Targeted Next-Gen BisulﬁteSequencing Now Offered as a Service

11 NEW: Long-term Staining of Membranes inLive or Fixed Cells & Tissue

6 NEW: Multiplex Transcription Factor Assays
for Use with Luminex® Instruments

12 Prevent Wasted Time and Effort with our
ChIP Antibody Validation Services

8 Arrays, Antibodies and Kits for the Study ofHistones and their Modiﬁcations

12 Monitor Binding of Active Transcription
Factors with TransAM® Assays

Chromatin Immunoprecipitation

NEW: ChIP-IT® High Sensitivity Kit for Difﬁcult-to-ChIP Transcription Factors

Works with limited sample material
Due to the high sensitivity of this ChIP
kit, immunoprecipitation reactions canbe performed from as little as 1,000 cells
for highly abundant target proteins, suchas histones, and as little as 50,000 cells
for low abundance proteins (Figure 2).

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9ecf[j_jeh:Figure 1: The ChIP-IT High Sensitivity Kit detects low abundance target proteins better than the competition.
Chromatin was prepared according to the recommendations for each manufacturer’s ChIP kit from 1.5 x 106 MCF-7 cells
per assay. ChIP was performed with the optimal amount of chromatin suggested in each manufacturer’s protocol using anantibody for the low abundance nuclear co-activator 2 (NCOA2) protein and a negative control IgG. Following enrichment,
qPCR was performed using the ChIP-IT qPCR Analysis Kit (Catalog No. 53029, see page 3) to normalize the data forchromatin amounts and ChIP volume, as this enables a direct comparison of the competitor kit results. While NCOA2 isconsidered a difficult antibody for ChIP, NCOA2 binding was detected at the estrogen-related receptor alpha (ESRRA)
promoter when ChIP was performed using ChIP-IT High Sensitivity; the enrichment was approximately 20-fold higher
than the negative control primer set and the IgG. When using competitor kits, NCOA2 was either not enriched at all(Competitors M & I), or there was non-specific binding by the IgG and high background for the negative control primers(Competitor D). Data represents triplicate qPCR values and is expressed as binding events detected per 1,000 cells.
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The ChIP-IT High Sensitivity advantage• Sensitivity – optimized to obtainhigh signals and low background forthe detection of low abundanceproteins or when using antibodies
with low binding afﬁnities• Low Cell ChIP – enrichmentfrom as little as 1,000 cells per
immunoprecipitation reaction• Ease of Use – ﬁltration columns
make the wash steps quick and easy
• Consistency – extensive testingwith multiple sample types andantibodies has validated the kit’s
performance in ChIP-Seq and qPCR('#'''

ChIP-IT High Sensitivity uses speciallyformulated buffers for chromatin preparation from cultured cells or fresh or
frozen tissue. Low background protein G
agarose beads and an antibody blocker
are used to minimize any non-speciﬁc
binding during the immunoprecipitation
reaction, enabling a higher degree ofsensitivity than other commercially available ChIP Kits (Figure 1). Filtration columns provide a fast, easy and consistentsolution for wash steps; DNA puriﬁcation reagents are included to complete
the workﬂow. Recommendations areincluded for the use of the high-quality
ChIP DNA in qPCR and ChIP-Seq.

*,

8_dZ_d][l[djiZ[j[Yj[Z%'"&&&Y[bbiWhy another ChIP Kit?
Active Motif has decided to expand its
ChIP-IT product offering to meet theneeds of those researchers studying low
abundance transcription factors, low
afﬁnity antibodies or using limited sample material. The ChIP-IT High Sensitivity
Kit is speciﬁcally designed to give higher
signal and reduced background, ensuringthat low abundance binding events aredetectable in downstream applications
such as ChIP-Seq, ChIP-chip and qPCR.

8_dZ_d][l[djiZ[j[Yj[Z%'"&&&Y[bbiAs the ﬁeld of epigenetics expands into the research areas of transcriptional regulation and stem cells, it has become
more important to identify methods to obtain accurate and reliable chromatin immunoprecipitation (ChIP) resultsfor difﬁcult-to-ChIP transcription factors and limited cell numbers. Active Motif’s new ChIP-IT® High Sensitivity Kit
is designed to meet this need by enabling you to enrich for high-quality ChIP DNA even when working with low
abundance transcription factors, low-afﬁnity antibodies or limited starting material. Not only is the ChIP-IT HighSensitivity Kit capable of enriching DNA from as little as 1,000 cells, but the kit has been successfully ChIP-Seq
validated with hundreds of samples and target antibodies.

9WjWbe]De$-'&&,

Figure 2: ChIP-IT High Sensitivity IP range of detection.
MCF-7 chromatin was prepared according the ChIP-IT
High Sensitivity manual using between 100,000 and
4 million cells. ChIP was performed using the number ofcells indicated with Histone H3K4me3 antibody and IgG.
Specific enrichment is detected in as few as 1,000 cells.

Product

FormatCatalog No.

ChIP-IT® High Sensitivity

16 rxns

53040

Toll Free — 1 877 222 9543

October 2012 • volume 13 • number 2

Chromatin Immunoprecipitation

NEW: qPCR Analysis Strategy to Normalize ChIP Data for Easier Interpretation
Traditional methods to analyze ChIP qPCR results usually rely on fold enrichment of ChIP DNA over the enriched
DNA from an IgG or non-speciﬁc antibody immunoprecipitation reaction. One disadvantage of this method is that
the IgG reaction is performed separately, so any enrichment seen in the IgG reaction may not be directly relevant towhat occurred in the ChIP reaction of interest. Another disadvantage is that this method also requires generating astandard curve for every primer set tested so as to determine the efﬁciency of each PCR primer pair. Finally, if different amounts of chromatin were used in the ChIP reactions, interpretation of results between samples or experiments
is extremely difﬁcult. To overcome these problems and simplify ChIP qPCR data analysis, Active Motif has developed
the ChIP-IT® qPCR Analysis Kit and its associated Excel-based ChIP-IT® qPCR Analysis spreadsheet.

The ChIP-IT qPCR Analysis Kit includes
DNA standards, a standard curve primer
pair, as well as control qPCR primers foruse with human and mouse samples.
The kit contains sufﬁcient reagents toperform ten qPCR standard curves.

www.activemotif.com

The recommendations are based on theuse of the ChIP-IT High Sensitivity Kit(Catalog No. 53040) to perform the chro-

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Data from the ChIP-IT qPCR Analysis Kit
is presented as binding events detected
per 1,000 cells. To see published data using this qPCR normalization strategy, or
to download the manual for an explanation of the calculations, please visit us atwww.activemotif.com/qPCRanalysis.

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matin immunoprecipitation reactions. If
another method is used to perform ChIP
that results in higher background levels
and lower sensitivity, the recommended
threshold levels may not apply.

8_dZ_d][l[djiZ[j[Yj[Z%'"&&&Y[bbiFollowing qPCR ampliﬁcation, the values
determined by the qPCR instrument are
copied into the ChIP-IT qPCR Analysis
spreadsheet. The spreadsheet performs
calculations to normalize the data withrespect to primer pair efﬁciency, theamount of chromatin in each reaction
and the resuspension volume of the
ChIP DNA, then generates a graph ofthe results. The spreadsheet templateis designed to accommodate data frommultiple 96-well PCR plates into a single
analysis, making it easier to compare
multiple sample types and experiments(see Figure 1 on page 2).

Interpreting the ChIP DNA quality
Recommendations for data interpretation are provided to evaluate the successof the ChIP reactions and the quality of
the ChIP DNA. Following these recommendations will provide conﬁdence that
the ChIP DNA is of a high quality before
proceeding with expensive downstream
applications such as ChIP-Seq.

8_dZ_d][l[djiZ[j[Yj[ZHow does it work?
The ChIP-IT qPCR Analysis Kit contains
known DNA standards to create a single
standard curve per 96-well PCR plate;this curve is used to determine ChIP DNA
quantity and to assess the efﬁciency ofmultiple PCR primer sets. ChIP DNA istested using a gene-speciﬁc primer of interest and the provided negative control
primer set. The negative control primer
serves as a measure of background in theactual ChIP reaction of interest, and is anindicator of the quality of the ChIP DNA.

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8:K9Gi`d\iJ\kFigure 1: The ChIP-IT qPCR Analysis Kit enables direct comparison of ChIP efﬁciency.
ChIP was performed using an RNA pol II antibody with the ChIP-IT High Sensitivity Kit. Different amounts of chromatin wereused in each ChIP reaction ranging from 3,000 cell equivalents to 150,000 cell equivalents. In the left graph, normalization was
performed independent of the starting amount of chromatin, taking only the primer efficiency and resuspension volume
into account. In the right graph, Active Motif’s ChIP-IT qPCR Analysis Kit was used to also normalize for the starting amountof chromatin. This data demonstrates the dynamic range of the ChIP-IT High Sensitivity Kit and the accuracy of thenormalization strategy in the ChIP-IT qPCR Analysis Kit.

Product

FormatCatalog No.

ChIP-IT® qPCR Analysis Kit

10 rxns53029

3

Nucleosome Occupancy & DNA Methylation

NEW: Enhanced Gene Regulation Analysis with NOMe-Seq
Active Motif’s NOMe-Seq assay is the ﬁrst commercially available kit to simultaneously interrogate the relationshipbetween nucleosome occupancy, transcription factor binding and DNA methylation within the same DNA molecule.
It provides high-resolution mapping of nucleosome occupancy that enables even subtle changes in nucleosome
position to be determined. By studying nucleosome occupancy in combination with DNA methylation at a speciﬁc
gene locus, NOMe-Seq provides researchers with a tool to better understand the different states of chromatin andtheir effects on gene regulation. To learn more, visit us at www.activemotif.com/nome-seq.
DEC[#I[gC[j^eZWhat is NOMe-Seq?
NOMe-Seq stands for Nucleosome
Occupancy and Methylome Sequencing.
It is a technique that was developed by
the Peter A. Jones lab at USC to simultaneously study nucleosome occupancy
and CpG methylation on the same DNA
strand. Traditional methods have only
been able to look individually at DNA
methylation (bisulﬁte sequencing) or at
nucleosome positioning (MNase-seq or
FAIRE-seq) without the ability to understand the relationship between the two.
In NOMe-Seq, ﬁxed chromatin is treatedwith a GpC DNA methyltransferase
enzyme to artiﬁcially methylate any GpC
dinucleotides that are not protected by
the binding of nucleosomes or proteinsto the DNA. Because GpC dinucleotides
are signiﬁcantly more abundant within
the genome than CpG dinucleotides,
their use allows for better resolution of
nucleosome positioning. Additionally, as
GpCs are not endogenously methylated
in mammalian genomes their artiﬁcial
methylation does not interfere with theability to establish an endogenous CpG
methylation proﬁle.
Following GpC methylation, the artiﬁcially methylated DNA is denatured
and treated with sodium bisulﬁte. This
converts unmethylated cytosines to uracil, while methylated cytosines remainunchanged. Gene-speciﬁc regions ofinterest are then PCR ampliﬁed, cloned
and the DNA is sequenced (Figure 1).

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Figure 1: The NOMe-Seq method.
NOMe-Seq works by formaldehyde fixation of cells to preserve nucleosome position as well as protein/DNA binding
interactions. Fragmented chromatin is enzymatically treated with a GpC methyltransferase to artificially methylate GpC
residues not protected by bound nucleosomes or proteins. Following reversal of cross-links and DNA purification, bisulfite
conversion is performed to identify the methylated cytosines on each DNA strand. For gene-specific analysis, the regions ofinterest are PCR amplified, cloned and sequenced. The GpC methylation profile will determine the nucleosome or protein
binding site positions, while the CpG methylation reveals the endogenous methylation pattern of the gene of interest.

The sequence results are then mapped
to compare the artiﬁcially methylated
GpC and the endogenously methylated
CpG residues. Regions of unmethylated
GpC residues that are 147 bp or larger
represent nucleosome occupancy,
which prevented GpC methylation by
the enzyme. Unmethylated regions of
10-80 bp indicate sites of transcription

factor binding. By combining this withthe sequence data of endogenous CpG
methylation, one can correlate simultaneous nucleosome and transcription
factor occupancy with DNA methylation all within the same DNA molecule.
NOMe-Seq can also be used in wholegenome experiments, as demonstrated
in a recent publication.

Product

FormatCatalog No.

NOMe-Seq

10 rxns

54000

Toll Free — 1 877 222 9543

October 2012 • volume 13 • number 2

Epigenetic Services

NEW: MethylCollector™ Ultra-Seq is Now Available as a Service
Active Motif’s popular MethylCollector Ultra Kit, which is used to speciﬁcally enrich for methylated DNA fromfragmented genomic DNA, is now offered as an end-to-end, full service assay by Active Motif’s Epigenetic Services.
Customers can submit their cells or DNA and receive fully analyzed genome-wide methylation data in just weeks.
MethylCollector Ultra, based on thepatented MIRA (Methylated CpG Island
Recovery Assay) technology, offers thefastest, most efﬁcient methylated DNA

enrichment method on the market.
Enrichment is achieved from as little as
1 µg of DNA using a puriﬁed methylated
DNA binding complex of MBD2b andC[j^ob9ebb[YjehKbjhW

9ecf[j_jehC

THRAP3

Clorf113
p34.3

36,760,000

36,780,000

36,800,000

36,820,000

STK40

36,840,000

LSM10

Figure 1: MethylCollector Ultra is more sensitive than other MBD-based enrichment methods.
To ensure that we provide scientists with the best possible results, the performance of MethylCollector Ultra wascompared to that of an alternative MBD-Capture kit. Methylated DNA was isolated from 1 µg of human PBMC DNA using
both enrichment kits, then the enriched DNA was sequenced using the Illumina HiSeq platform. Regions of enrichmentfrom a 1.2 Mb region of the genome are shown above. The MethylCollector Ultra technology detected more methylated
regions and the peaks were of greater intensity than those detected by the competitor’s kit.

MBD3L1. This is followed by magnetic
capture of double-stranded DNA that iscompatible with Next-Gen Sequencing
library preparation protocols.
The New MIRA-Seq Service includes• DNA isolation from cells or tissues• Methylated DNA enrichment
using MethylCollector Ultra• qPCR analysis of positive andnegative control sites• Next-Gen Library generation• Sequencing of at least 30 million
tags using the Illumina HiSeq
platform• Analysis: mapping, peak calling,visualization ﬁles and Excel output
For more complete details on this andother services offered, please visit us atwww.activemotif.com/services.

NEW: Targeted Next-Gen Bisulﬁte Sequencing Now Offered as a Service

www.activemotif.com

Next-Gen Bisulﬁte Sequencing Service
combines many bisulﬁte-converted
amplicons from multiple samples intoa single Next-Gen sequencing run. It
is ideal for use as a follow-up assay to
MethylCollector Ultra, providing single
base pair resolution of DNA methylation
across many cell populations.
Active Motif Epigenetic Services• Experience – over 1500 genomewide data sets generated• Quality – QC steps ensure highquality whole-genome data
• Support – All services include
bioinformatics analysis
For more complete details, please
visit www.activemotif.com/services.

-('

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Identify differentially methylated
regions of DNA
The identiﬁcation of differential methylation across multiple samples is an
important strategy for determining theunderlying mechanisms of development
and disease. However, while a complete
understanding of these mechanisms
may require base pair resolution of DNA
methylation, the current costs for wholegenome bisulﬁte sequencing remainprohibitive. A great alternative to wholegenome bisulﬁte sequencing is to use
MethylCollector Ultra to identify regionsof differential methylation followed bytargeted bisulﬁte sequencing of such
identiﬁed regions in a larger populationof samples. Active Motif’s Targeted

(*

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:D7C[j^obWj_ed

Figure 1: Targeted Next-Gen Bisulﬁte Sequencing.
MethylCollector Ultra-Seq was performed on 3 samples
of interest to identify differentially methylated regions.
One identified region was used for Targeted Next-Gen
Bisulfite Sequencing on a broader population of 9 samples. This heat map shows the single base pair resolutionof the 72 CpGs in this region across the 9 samples.

5

Multiplex Transcription Factor Assays

NEW: Multiplex Transcription Factor Assays for Use with Luminex® Instruments
Active Motif is pleased to announce the release of its new Transcription Factor Multiplex Assays to study NFκB and
AP-1 transcription factor binding events using either the Luminex® 200™ or MAGPIX® instruments. The assays workas pull-down reactions of biotinylated DNA probe-transcription factor complexes with transcription factor-speciﬁc
antibodies conjugated to ﬂuorescently labeled magnetic beads. Because each antibody-conjugated bead contains a
different ﬂuorescent label, the assay can be multiplexed to study the entire transcription factor family within a single
well of the assay. Alternatively, individual family members can be assayed on their own as a singleplex.
Bead-based transcription factor assays
The Transcription Factor Multiplex
assays work as pull-down reactions
where cell lysates are combined with abiotinylated double-stranded consensus
binding sequence for the transcription
factor family of interest. Fluorescently
labeled magnetic beads, which havebeen conjugated to antibodies thattarget the individual transcription factor
family members, are used to capture
the transcription factor-bound DNA.
Streptavidin-phycoerythrin is used tobind the biotinylated oligo and determine the magnitude of signal (Figure 1).
Because each antibody-conjugated bead
contains a different ﬂuorescent label,
antibodies for multiple family members
can be added to the same sample formultiplexing. The readout from thestreptavidin-phycoerythrin is proportional to the amount of transcription
factor binding. The data can be used
to compare transcription factor
binding activity among the various
family members.
The Luminex® Principle
The Luminex Principle is based on theuse of tiny, 6.4 micron superparamagnetic beads, each containing their own
distinct dye ratio. Inside the Luminex
200™ instrument, the beads are individually passed through a small shaft where alight source excites the internal dye thatis used to identify each bead. Based on
the dye ratios, each bead will exhibit a6

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Figure 1: Transcription Factor Multiplex Assays are bead-based assays to measure TF-DNA binding activity.

unique ﬂuorescence that can be identiﬁed with a unique bead number. A second wavelength is used to determine themagnitude of the streptavidin-phycoerythrin signal (Figure 2). A minimum of
100 beads are read for each well of theassay. The Luminex xPONENT software
program will provide a real-time readoutof signal as median ﬂuorescent intensity(MFI) (Figure 3, page 7).

Figure 2: The Luminex 200™ instrument.
The Luminex 200 instrument uses two distinct wavelengths to analyze individual beads as they pass throughthe machine for bead identity and phycoerythrin signal.

What’s in the box?
The Transcription Factor Multiplex
assays include a 96-well assay plate, abiotinylated oligonucleotide containing
the consensus binding sequence for thetranscription factor family of interest,
and wild-type and mutant competitor
oligos to conﬁrm the speciﬁcity of thereactions. Streptavidin-phycoerythrin,
assay buffers and a positive control celllysate are also included.
The transcription factor antibody-conjugated beads are purchased separately.
This allows the assay to be customized
to ﬁt the individual needs of each researcher. All antibody-conjugated beads
have been conﬁrmed to work when multiplexed without any negative effects on
the assay results. Simply select the beadsets of interest and the Transcription
Factor Multiplex Buffer Kit and you will
have all the reagents needed to performthe assay.

Toll Free — 1 877 222 9543

October 2012 • volume 13 • number 2

Transcription Factor NFκB
NFκB is a widely studied transcription
factor family due to its implications in
the regulation of genes that control
inﬂammation, cell proliferation and cell
survival. The NFκB family is comprisedof a mixture of homodimeric and heterodimeric subunits that are membersof the structurally related Rel family oftranscription factors. NFκB p65 (RelA),
RelB and c-Rel contain a transactivation
domain that is required for translocation of active NFκB complexes into the
nucleus where they are able to bindDNA. The p50 and p52 subunits do notcontain the transactivation domain andmust form heterodimers. Active Motifcurrently offers antibody-conjugatedbeads for NFκB RelB and NFκB p52 subunits, with other NFκB family members
to become available soon.
TF Multiplex advantages• Reduction in both costs & labor• Simultaneously analyze different
subunits of the same transcription
factor family in the same sample• Individual bead sets enable theassay to be customized

www.activemotif.com<ei8I_d]b[fb[n7iiWo

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Transcription Factor AP-1
AP-1 is a transcription factor family
consisting of a mixture of homodimericand heterodimeric protein complexes
derived from the Fos and Jun families.
The AP-1 proteins play a role in theexpression of genes involved in regulation of differentiation, proliferation andapoptosis. AP-1 expression is induced bymultiple stimuli, including the presenceof serum, growth factors, phorbol esters
and oncogenes. Phosphorylation of AP-1
family members by kinases is required
for transactivation activity. Once theAP-1 family members have translocatedinto the nucleus they are able to bindDNA. Active Motif offers antibody-conjugated beads for each of the following
AP-1 family members: c-Fos, FosB, Fra-1,
JunB and JunD.

Multiplex Transcription Factor Assays

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Figure 3: Transcription factor AP-1 studied as singleplex or multiplex assay.
The Transcription Factor Multiplex Kit – AP-1 was used to study the DNA binding activity of various members of the AP-1
transcription factor family. The top images show singleplex data for FosB and JunB assays run individually. The positive
control lysate, K-562 nuclear extract treated with TPA, is tested in the absence or presence of the provided competitor
oligos. The wild-type oligo shares the same consensus sequence as the biotinylated consensus oligonucleotide. Therefore, it
competes for binding with the transcription factor, inhibiting the ability of the transcription factor to bind the biotinylatedconsensus oligonucleotide. The mutant oligonucleotide has a modified consensus sequence that is not recognized by thetranscription factor and therefore does not compete for transcription factor binding. The use of the wild-type and mutant
oligos within the assay confirm the specificity of the assay. The bottom image shows a multiplex assay in which multiple
transcription factor antibody-conjugated beads were combined into a single well of the assay to study the transcription
factor activity of the various AP-1 family members within the same sample. A comparison of the top and bottom images
shows that multiplexing does not have any negative effects on the assay results.

To learn more about the new Transcription Factor Multiplex Assays
for use with Luminex instruments, please call or visit us atwww.activemotif.com/luminex.
Product

FormatCatalog No.

Transcription Factor Multiplex Kit – AP-1

100 rxns

33100

c-Fos-conjugated beads

48 rxns

33101

FosB-conjugated beads

48 rxns

33102

Fra-1-conjugated beads

48 rxns

33103

JunB-conjugated beads

48 rxns

33104

JunD-conjugated beads

48 rxns

33105

Transcription Factor Multiplex Kit – NFκB

100 rxns

33110

RelB-conjugated beads

48 rxns

33111

NFκB p52-conjugated beads

48 rxns33112

7

Histone Modification Products

Arrays, Antibodies and Kits for the Study of Histones and their Modiﬁcations
Histone modiﬁcations such as acetylation, phosphorylation and methylation at speciﬁc amino acid residues on
the histone globular domain and the N-terminal tails have been shown to inﬂuence and regulate transcription,
chromosome packaging and DNA damage repair. Due to the importance of histone modiﬁcations in regulating
chromatin structure and disease, Active Motif has developed a variety of products to simplify histone analysis
including MODiﬁed™ Histone Peptide Arrays and a large number of ELISAs, kits, antibodies and proteins (page 9).

Verify antibody speciﬁcity ﬁrst
MODiﬁed Arrays are ideal to screen
histone modiﬁcation antibodies for nonspeciﬁc cross-reactivity with alternate
modiﬁcations. Using a simple protocolthat works like a Western blot, an imageof the array is captured and the antibody
binding is quantiﬁed by the provided
software (Figure 1). This valuable datalets you know if your antibody is speciﬁc
or not before investing your resources
using it in downstream assays.
Study proteins and enzymes thatinteract with histones
In addition to screening antibody
speciﬁcity, MODiﬁed Arrays can be used
to study proteins and enzymes thatinteract with and/or modify histones
as well as their inhibitors. The activity(+'>_ijed[>)A/c[)f7X()'

If[Y_\_Y_jo\WYjeh

Unique array to screen interactions
The MODiﬁed™ Histone Peptide Array*can be used to screen antibodies, proteins and enzymes for interactions withhistones and their post-translational
modiﬁcations. Each array contains 384
different combinations of acetylation,
phosphorylation, methylation and citrullination modiﬁcations on the N-terminal
tails of histones H2A, H2B, H3 and H4.
Because each peptide can contain up
to 4 different modiﬁcations, the arraycan be used to study not only individual
modiﬁcations, but also to determine
if neighboring modiﬁcations alter site
recognition and binding.

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of methyltransferases, demethylases,
acetyltransferases, etc. can be assayed at
speciﬁc histone modiﬁcations simply by
incubating the compound(s) of intereston the array followed by detection witha speciﬁc antibody (Figure 2).
An entire collection of products forstudying histone modiﬁcations
Active Motif also offers Histone Modiﬁcation ELISAs for histone quantiﬁcation,
kits for purifying core histones, assembling chromatin in vitro, and for studying
HATs, HDACs and demethylases. In addition, we offer over 160 highly speciﬁc
histone modiﬁcation antibodies thathave been validated using the MODiﬁed
Array and/or dot blots, as well as a large
collection of recombinant histone
proteins (page 9) and histone modifying
enzymes. For complete details, please
visit us at www.activemotif.com/histone.

Figure 1: Peptide array analysis
conﬁrms the speciﬁcity of H3K9me3
pAb for its intended modiﬁcation.
Histone H3 trimethyl Lys9 antibody(Catalog No. 39161) was applied at adilution of 1:2,000 to the MODified
Histone Peptide Array and scannedwith Array Analyse Software 15. The
results plotted in the adjacent graphshow specificity data for the ten mostreactive peptides, demonstrating that
this antibody is specific for H3K9me3,
with little binding at other sites.

7

8

9

Figure 2: Detection of G9a histone methyltransferase
activity using the MODiﬁed Histone Peptide Array.
MODified Histone Peptide Arrays were treated withA) 25 µM G9a methyltransferase, B) 25 µM G9a mutant
methyltransferase or C) a no enzyme control overnightin the presence of 1 mM AdoMet. The arrays were thenlabeled with a Histone H3 dimethyl Lys9 antibody. Novel
methylation sites were observed on the array treatedwith wild-type G9a histone methyltransferase, showing
the activity of G9a enzyme on the peptide substrate.

Product

FormatCatalog No.

MODiﬁed™ Histone Peptide Array

1 array
5 arrays

13001
13005

*CelluSpots™ arrays are manufactured under license by INTAVIS Bioanalytical Instruments AG and sold through Active Motif as MODiﬁed™ Histone Peptide Arrays

8

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October 2012 • volume 13 • number 2

Recombinant Histones and Bromodomains

Recombinant Proteins to Analyze Histone Post-translational Modiﬁcations
Understanding the implications of histone post-translational modiﬁcations as they relate to gene regulation andchromatin remodeling is critical to determining how these modiﬁcations affect or contribute to development
and disease. Active Motif’s expanded line of recombinant proteins for use in epigenetics research now includes
biotinylated histones and recombinant bromodomain proteins to speciﬁcally meet the needs of researchersinterested in the analysis of histone modiﬁcations. For complete details, visit us at www.activemotif.com/info
to download our Histone Analysis Products brochure or request a copy of by mail.

Recombinant Histone Proteins to Analyze Site-speciﬁc Modiﬁcations
Recombinant histone proteins withsite-speciﬁc modiﬁcations
To broaden your choice of histone analysis tools, Active Motif offers unmodiﬁed
recombinant histones as well as recombinant histones that include site- anddegree-speciﬁc methylation, acetylation,and phosphorylation modiﬁcations. Each
recombinant protein is prepared using
either our patented Expressed Protein
Ligation (EPL) or Methylated Lysine
Analog (MLA) technologies.

New biotinylated H3 proteins
To expand the versatility of our recombinant histones, Active Motif now
offers biotinylated versions of unmodiﬁed Recombinant Histone H3 as well as
Recombinant H3 with methyl modiﬁcations at Lys4 and Lys9. The biotinylated
recombinant histones are engineeredusing the patented EPL technology thatenables the incorporation of site-speciﬁc
modiﬁcations to the histone tail while
preserving its native structure. The

Product

FormatCatalog No.

Recombinant Histone H3 biotinylated

25 µg

31271

Recombinant Histone H3K4me1 biotinylated (EPL)

25 µg

31284

Recombinant Histone H3K4me2 biotinylated (EPL)

25 µg

31283

Recombinant Histone H3K4me3 biotinylated (EPL)

25 µg

31282

Recombinant Histone H3K9me1 biotinylated (EPL)

25 µg

31286

Recombinant Histone H3K9me3 biotinylated (EPL)

25 µg

31285

modiﬁed tail is ligated to the histone
globular domain using a method known
as “natural chemical ligation” that
preserves the native peptide bond. The
recombinant histones are biotinylated
at the N-terminus and, as a result of EPL
synthesis, more closely mimic “natural”
histones, making them ideally suited
as controls for the analysis of histone
modiﬁcations, as substrates for histone
modiﬁcation enzymes, and for use inin vitro chromatin assembly.

For an up-to-date list of themore than 40 recombinanthistone proteins currently
available, please visit us atwww.activemotif.com/recombhis.

NEW: Recombinant Bromodomain Proteins to Study Bromodomain Function
What are bromodomains?
Active Motif has expanded its line ofrecombinant proteins to include 15 newrecombinant bromodomain (BRD) proteins. BRDs function as “readers” of thehistone code. They are the only protein
domains capable of speciﬁcally recognizing and binding to acetylated lysine residues on histone tails. BRDs regulate gene
expression and chromatin remodeling
events by recruiting associated protein
complexes to acetylated chromatin.

www.activemotif.com

Why use recombinant bromodomains?
Aberrant acetylation has been implicated as the cause of various diseases,
including neurological disorders, cancer
and inﬂammation. Therefore, BRD-containing proteins have emerged alongside
HATs and HDACs as therapeutic targets
for drug development and discovery.
To aid in studies of BRD function,
Active Motif now offers recombinantbromodomain(s) of several common
BRD-containing protein families, includ-ing HATs, transcriptional coactivators
and BET family proteins, to name afew. These highly puriﬁed recombinantbromodomains are suitable for use invarious applications, including studies of
enzyme kinetics, inhibitor screenings andselectivity proﬁling.
For an up-to-date list of our available
recombinant bromodomains andcomplete details, please visit us atwww.activemotif.com/brd.

9

Reagents for Fluorescence

Reagents for High-quality Fluorescence Microscopy
To produce superior results with our primary antibodies in immunoﬂuorescence (IF) experiments, Active Motif has
developed a variety of high-quality reagents including Chromeo™ secondary antibody conjugates and MAX Stain™
Immunoﬂuorescence Tools. These optimized reagents can be used in a variety of ﬂuorescence microscopy applications,even in demanding high-resolution ﬂuorescence microscopy (e.g. STED and GSDIM). We use these reagents in ourlabs to help us develop our novel assays and to evaluate our antibodies in a consistent and reliable manner.
Fluorescent secondary antibodies
Active Motif’s goat anti-mouse and goatanti-rabbit secondary antibodies areconjugated to a number of high-quality
dyes, including Chromeo™ ﬂuorescentdyes (Table 1). Our optimized conjugation method, coupled with subsequentpuriﬁcation of the conjugate, makes ourFluorescent Dye

Absorption

ﬂuorescent secondaries brighter thanother commercially available conjugatesand lowers the background in many
applications. Active Motif’s antibody
conjugates have been validated for use
in a variety of applications, such as ﬂow
cytometry, and wideﬁeld, confocal andhigh-resolution ﬂuorescence microscopy.
Emission

Replaces

Chromeo™ 488

498 nm524 nm

FITC, Alexa 488*

Chromeo™ 494

489 nm624 nmuniqueChromeo™ 505

514 nm

530 nmOregon Green derivatives

Chromeo™ 546

550 nm

567 nmCy3, Alexa 546*

Chromeo™ 642

647 nm

666 nmCy5, Alexa 647*

Figure 1: Chromeo ﬂuorescent secondaries and
MAX Stain Tools provide bright, high-quality images.
HeLa cells were stained with alpha Tubulin mouse mAb(Catalog No. 39527) and Chromeo 488 Goat anti-mouse
IgG (Catalog No. 15031). The nuclei have been counterstained with DAPI. In all steps of slide preparation,
MAX Stain Immunofluorescence Tools have been used.

Table 1: Fluorescent properties of dyes when conjugated to secondary antibodies.
To see a complete list of fluorescent secondary antibody conjugates, please visit www.activemotif.com/fluorabs.

Better slide prep for the best IF images
MAX Stain™ Immunoﬂuorescence Tools
will help you to produce high-quality IF
images every time by providing you witha complete set of optimized reagentsfor slide preparation. The MAXblock and
MAXwash reagents use non-mammalian
agents to effectively block non-speciﬁc
antibody binding. The MAXpack
Immunostaining Media Kit includes one
each of the MAXblock, MAXbind andMAXwash reagents. For details, visit us
at www.activemotif.com/maxstain.

Advantages of MAX Stain reagents• MAXblock™ Blocking Medium –effectively blocks non-speciﬁc
antibody binding without reducing
signal intensity or speciﬁcity• MAXbind™ Staining Medium –increases the speciﬁcity of antibody
binding as well as the ﬂuorescentintensity of your IF experiments• MAXwash™ Washing Medium –eliminates non-speciﬁc binding ofprimary and secondary antibodies

Product

FormatCatalog No.

MAXblock™ Blocking Medium

150 ml

15252

MAXbind™ Staining Medium

250 ml

15253

MAXwash™ Washing Medium

1000 ml

15254

MAXpack™ Immunostaining Media Kit

1 kit

15251

* Alexa Fluor dyes are a registered trademark of Life Technologies™.

10

Figure 2: Chromeo ﬂuorescent secondaries insuper-resolution GSDIM microscopy.
Tubulin was stained with a primary monoclonal mouse
antibody and with Chromeo 546 Goat anti-mouse IgG(Catalog No. 15033) secondary antibody. Image courtesyof Leica Microsystems, Germany.

Why use Chromeo™ conjugatedsecondary antibodies?
• Brightness – high ﬂuorescentintensity improves sensitivity• Limited photobleaching – enables
multiple exposures and increased
exposure times• Flexibility – conjugates work
under multiple ﬁxation conditions• Specificity – low ﬂuorescentbackgroundToll Free — 1 877 222 9543

October 2012 • volume 13 • number 2

Cell and Organelle Stains

NEW: Long-term Staining of Membranes in Live or Fixed Cells & Tissue
The Chromeo™ LT Live Cell Membrane Staining Kit* features a cell permeable ﬂuorescent complex that selectively
stains lipid structures like plasma and internal membranes with high speciﬁcity. Such ﬂuorescent stains speciﬁcallyintercalate into membranes so they can be used not only to label the different cellular compartments, but also as aversatile and powerful tool for long-term (LT) live cell imaging. The Chromeo LT Live Cell Membrane Staining Kit canbe used to monitor cell number, cell proliferation and the effect on cell growth or death in response to treatment bydrugs, activators, inhibitors or radiation. In addition, it can serve as a classical counter-stain in immunoﬂuorescence
experiments to visualize lipid structures in live or ﬁxed cells as well as in FFPE tissue sections.
The Chromeo LT Live Cell Membrane
Staining Kit utilizes a proprietary, watersoluble ﬂuorescent complex that rapidly
diffuses into live or ﬁxed cells and tissue.
The stain accumulates in lipid structures
such as plasma and internal membranes,
lysosomes or lipid bodies, allowing them
to be speciﬁcally labeled and imaged
with low background. Its afﬁnity for
and retention in lipid structures, and its
stability towards photobleaching, makeit possible to monitor cells over a periodof several days.

Why use the Chromeo LT Live Cell
Membrane Stain?
• Water-soluble ﬂuorescent stain forlive cells or Formalin-Fixed, ParafﬁnEmbedded (FFPE) cells and tissue• Low toxicity• Easy to use• Excitable at 488 nm• Use FITC ﬁlter sets
• Long-term labeling experiments

In addition to the Chromeo LT Membrane Stain, Active Motif offers several otherﬂuorescent cell and organelle stains (Table 1). For more complete information anda vast amount of application data, please visit www.activemotif.com/stains.
Cell and Organelle Stain

Live cells Fixed cells Long-term staining

Excitation

Emission

Chromeo™ LT Live Cell
Membrane Staining Kit

√

√

√

420 - 490 nm

510 nmChromeo™ Live Cell
Mitochondrial Stain

√

√

√

470 - 550 nm

610 nmChromeo™ Red Fluorescent
Fixed Cell Staining Kit

–

√

–

520 - 550 nm

627 nmLavaCell™ Live Cell
Membrane Staining

√

√

√**

405, 488, 532 nm

610 nmFigure 1: Staining of live NRK cells with the Chromeo
LT Live Cell Membrane Stain.
NRK cells were plated in 3 cm cell culture dishes andwashed once with pre-warmed PBS before adding 30 nM
Chromeo LT Live Cell Membrane staining solution. Afterincubation for 10 minutes at 37°C, cells were washedonce with pre-warmed PBS and imaged in PBS with a
Nikon CLSM (60x objective).

** While LavaCell is an excellent tool for long-term studies, the dye must be kept in the culture medium during theentire experiment.
Table 1: Fluorescent properties and performance overview of Active Motif’s organelle-speciﬁc dyes.

Product

FormatCatalog No.

Chromeo™ LT Live Cell Membrane Staining Kit

1 kit

15007

Chromeo™ Live Cell Mitochondrial Stain

1 kit

15005

Chromeo™ Red Fluorescent Fixed Cell Staining Kit

1 kit

15006

LavaCell™ Live Cell Membrane Staining

200 µg

15004

Figure 2: Staining of Formalin-Fixed ParafﬁnEmbedded (FFPE) epithelial carcinoma with
the Chromeo LT Live Cell Membrane Stain.

* The Chromeo™ LT Live Cell Membrane Stain is manufactured under license by OnkoTec GmbH.

www.activemotif.com

11

ChIP Services & Transcription Factor ELISAs

Prevent Wasted Time and Effort with our ChIP Antibody Validation Services
Finding an antibody that performs well in ChIP is one of the biggest challenges when initiating a ChIP project.
Traditional ChIP antibody validation using qPCR can be akin to “ﬁnding a needle in a haystack”. It requires substantial
time searching the literature for good binding sites in an appropriate cell type and stimulation conditions, and canbe inconclusive if the incorrect binding sites are interrogated. That is why, as part of our broader ChIP-Seq service,
Active Motif offers ChIP antibody validation services by ChIP-Seq and ChIP-chip (Diagram 1).
Antibody validation by ChIP-Seq
ChIP-Seq antibody validation isa small scale, ChIP-Seq reaction.
• More robust and moresensitive than ChIP-chip• Sequencing performedon an Illumina MiSeq• 3-4 week turnaround• Recommended when the
antibody target is expected
to be challenging

Antibody validation by ChIP-chip
ChIP-chip antibody validation isbased on hybridization to an array.
• Less expensive than ChIP-Seq• Hybridization to a single
Affymetrix tiling array• 2-3 week turnaround• Recommended whenantibody performance is
expected to be robust

Active Motif Epigenetic Services has over 8 years of experience
providing end-to-end ChIP services. We have processed over 10,000
ChIP samples, including over 4,000 ChIP-on-chip tiling arrays and over
1,000 ChIP-Seq reactions. To avoid wasting time and effort, start yournext project off right by utilizing our expertise to ChIP validate yourantibody. For details, please visit www.activemotif.com/services.

9^?F7dj_XeZoLWb_ZWj_edIj[fi' :ljkfd\ij\e[j8Zk`m\Dfk`]]`o\[Z\ccjfi]ifq\ek`jjl\%( 8Zk`m\Dfk`]gi\gXi\j
Z_ifdXk`eXe[jfe`ZXk\j%) 8Zk`m\Dfk`]g\i]fidj
k_\:_@Gi\XZk`fej%

9^?F#I[g9^?F#Y^_f* K_\:_@G;E8`jlj\[kf
ZfejkilZk:_@G$J\hc`YiXi`\j%* K_\:_@G;E8`jXdgc`]`\[#]iX^d\ek\[Xe[cXY\c\[%+ K_\c`YiXi`\jXi\j\hl\eZ\[lj`e^k_\@ccld`eXD`J\h%+ K_\cXY\c\[:_@G;E8`j_pYi`[`q\[kfk`c`e^XiiXpj%, K_\[XkX`jXeXcpq\[Xe[]`c\jXi\[\c`m\i\[kfk_\Zljkfd\i%Diagram 1: Active Motif performs all steps of the ChIP experiment from chromatin
preparation to data analysis, and provides an evaluation of the antibody’s performance.

Monitor Binding of Active Transcription Factors with TransAM® Assays
TransAM® Kits are a simple and efﬁcient, non-radioactive alternative to gelshift assays that can detect small changes
in transcription factor binding. TransAM Family Kits enable you to measure the activity of entire families of transcription factors in a single experiment. TransAM is by far the most referenced transcription factor DNA-binding assay,with over 800 literature citations. In addition, Active Motif offers the largest selection of transcription factor targets.
TransAM advantages• Detects only active transcription
factor-DNA binding events• Isoform specific detection• Works on all sample types
including cell lines, primaryisolates and tissues• Quantitative results in lessthan 3 hours
To see the complete list of all 48 TransAMKits, visit www.activemotif.com/transam.

12

Add nuclear extract
Nuclear extractcontaining activatedtranscription factor

Oligonucleotide
coated plate

Add primary antibody
1 hr.

Add developing
and stop solution

Add anti-IgG
HRP conjugate
1 hr.
1 hr.

Figure 1: Flow chart of the TransAM process.
Activated transcription factor in the sample binds to the immobilized oligo, containing the transcription factor consensusbinding site, in each well. Incubation with the supplied primary and secondary antibodies and addition of developing
solutions provides a specific, quantitative readout of activated transcription factor.

Toll Free — 1 877 222 9543