FOCUS OF THIS ISSUE

Histones:
Kits, ELISAs &Antibodies
Designed to
Unravel the
Histone Code

2 NEW: Novel Peptide Array Simpliﬁes
Histone Modiﬁcation Screening

8 Simple, Effective Monitoring of
Phosphorylation Using In-cell Detection

3 NEW: Easy Analysis of
Deubiquitinating Enzymes (DUBs)

8 Detect Active Transcription Factors
with TransAM™ Assays

3 NEW: First Commercially Available
Recombinant Acetylated Histones

9 Antibodies for the Study of
Polycomb Group Proteins

4 NEW: 5-Hydroxymethylcytosine – Newest
Epigenetic Mark, and 1st Antibody for Its Study

9 ChIP-validated Histone Monoclonal
Antibodies Ensure Your Success

5 NEW: Improved Format for
Methylated CpG Island Enrichment

10 NEW: Screen Histone PTMs with aFast, Sensitive ELISA

5 Speciﬁcally Detect Unmethylated CpG Islands

11 Using Histone and Histone Modiﬁcations
as Biomarkers for Cancer

6 Choose the ChIP Kit That is Best forYour Intended Application

Modified Histone Peptide Array

NEW: Novel Peptide Array Simpliﬁes Histone Modiﬁcation Screening
The importance of antibodies as research tools in epigenetic and chromatin analysis requires careful screening toensure accurate speciﬁcity against post-translational modiﬁcations, such as acetylation, methylation, phosphorylationand citrullination. To help overcome the obstacles of antibody characterization, Active Motif is pleased to introduce
the MODiﬁed™ Histone Peptide Array, a novel peptide array that enables you to screen 384 different histone
modification combinations, comprising 59 post-translational modiﬁcations, in a single experiment.

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Active Motif also offers antibodies forhistones and histone modiﬁcations. For
more information, please call or visit us
at www.activemotif.com/hismods.

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MODiﬁed Histone Array advantages• Histone specific – unique arraypanel speciﬁc for histones andhistone modiﬁcations• Multiple combinations – peptides
contain up to four modiﬁcation
combinations each
• Detects like a Western blot –works with either ECL-based orcolorimetric detection

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Better antibody characterization
With the increasing use of antibodies
as a research tool in techniques suchas ChIP, ChIP-seq, ChIP-chip and IF, it
is critical to assess antibody speciﬁcity
to ensure accurate data analysis. Active
Motif’s MODiﬁed Histone Peptide Array
offers a better way to screen antibodies
for cross reactivity. Each peptide array
contains 384 different histone modiﬁcation combinations, including up to four
separate modiﬁcations on the same
peptide. This extensive coverage ofhistone modiﬁcations will enable you to
study not only individual sites, but also
to determine the effects of neighboring
modiﬁcations on antibody recognition
and binding. MODiﬁed Histone Peptide
Arrays screen 59 acetylation, methylation, phosphorylation and citrullination
modiﬁcations on the N-terminal tails ofhistones H2A, H2B, H3 and H4.

Figure 1: The MODiﬁed Histone Peptide Array was used to evaluate the speciﬁcity of two different histone antibodies.
Active Motif’s Histone H3 dimethyl Lys9 mAb (Catalog No. 39683, page 9) was compared with a Histone H3 trimethyl Lys9
mAb. Each antibody was used at the recommended Western blot dilution factor. An anti-mouse HRP secondary antibody
was used at 1:2500 dilution, followed by ECL detection and image capture with CCD camera. Spot densitometry was used
to analyze intensity. Results were compared with the provided reference grid to determine the associated peptide content
and graphed as a fold intensity to represent the specificity factor. A) Camera image of Active Motif’s H3K9me2 mAb at
1 µg/µl dilution. B) Results indicate that Active Motif’s Histone H3 dimethyl Lys9 mAb has very little cross reactivity withother histone modifications. C) Camera image of H3K9me3 mAb at 1:1,000 dilution. D) Results indicate that the Histone H3
trimethyl Lys9 mAb antibody has a high degree of cross reactivity with other histone modifications.

How does it work?
To generate MODiﬁed Arrays, Active
Motif synthesizes a series of 19mer
histone H2A, H2B, H3 and H4 peptides,
each of which may contain as many asfour modiﬁcations. These are spottedin duplicate onto a glass slide, generating an array with a total of 384 unique
histone modiﬁcation combinations.
Following overnight blocking, the arrayis incubated with antibody for one hour,

washed and then incubated for one hourwith either a horseradish peroxidase or
alkaline phosphatase conjugated secondary antibody. Developing solution is thenadded and the image captured using ﬁlm
or a CCD camera; no special equipment
is needed. Software is used to analyze
the spot intensity; results are comparedwith the provided reference ﬁle showing
the grid pattern and associated peptide
content (Figure 1). For more details, go towww.activemotif.com/modiﬁed.

Product

FormatCatalog No.

MODiﬁed™ Histone Peptide Array

1 array
5 arrays

13001
13005

Get 10% off any single pack size MODiﬁed™ Histone Peptide Array.
Visit www.activemotif.com/promo for complete details.
2

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May 2010 • volume 11 • number 2

Deubiquitination Assay & Recombinant Histones

NEW: Easy Analysis of Deubiquitinating Enzymes (DUBs)
Measuring the activity of deubiquitinating enzymes (DUBs) can provide great insight into ubiquitin-dependent
metabolic pathways. The DUB-Detector™ Assay contains a ﬂuorescent ubiquitin substrate that when processed by
the cysteine protease class of DUBs releases a ﬂuorescent signal that is proportional to the amount of enzymatic
activity (Figure 1). This sensitive assay works with extracts or recombinant proteins and can be used to determine
the kinetics of the enzymatic reaction, or to screen for DUB inhibitors.
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Advantages of DUB-Detector• Fluorescent assay can be detected with an excitation
wavelength of 485 nm and an emission wavelength of 535 nm

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• Includes both a positive control extract and a DUB inhibitor

,'''
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• Fast procedure can be completed in less than 1 hour

)'

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-'

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Figure 1: Kinetic analysis of HeLa nuclear extract.
HeLa nuclear extracts were assayed at 0.313, 0.625 and1.25 µg per reaction in the presence or absence of
1 µM Inhibitor for 20 minutes. Following the incubation,
Fluorescent Substrate (100 nM) was added to each welland the fluorescent intensity was measured every two
minutes. Data shown are the results from duplicates.

• Great for either kinetic or end point analysis
Product

FormatCatalog No.

DUB-Detector™

1 x 96 rxns

40110

NEW: First Commercially Available Recombinant Acetylated Histones
Histone acetylation is a post-translational modiﬁcation that affects the nucleosome structure and therefore theability of transcription factors to access the DNA and regulate gene expression. To better support research on
histone acetylation, Active Motif is releasing its first acetylated recombinant histones.* These histones can beused as substrates for in vitro assays or as positive controls for acetylation detection.
Another ﬁrst for Active Motif
Active Motif was the ﬁrst company tooffer recombinant methylated histones
with site and degree speciﬁcity. Now
we have done it again with recombinant
acetylated histones. Our patent pending technology enables us to acetylate
the histone tail, without altering thenative peptide bonds. Our acetylated
recombinant histones are ideal for use as
substrates for in vitro assays or as positive controls for acetylation detection.
For complete information on all of ourrecombinant histones, please visit
www.activemotif.com/recombhis.

www.activemotif.com

Product

FormatRecombinant Histone H3 acetyl Lys9 NEW

25 µg

Recombinant Histone H3 acetyl Lys14 NEW

25 µg

Recombinant Histone H2A

50 µg

Recombinant Histone H2B

50 µg

Recombinant Histone H3 (C110A)

50 µg

Recombinant Histone H3 Lys4 (mono-, di-, or trimethyl)

50 µg

Recombinant Histone H3 Lys9 (mono-, di-, or trimethyl)

50 µg

Recombinant Histone H3 Lys27 (mono-, di-, or trimethyl)

50 µg

Recombinant Histone H3 Lys36 (mono-, di-, or trimethyl)

50 µg

Recombinant Histone H3 Lys79 (mono-, di-, or trimethyl)

50 µg

Recombinant Histone H4

50 µg

Recombinant Histone H4 Lys20 (mono-, di-, trimethyl)

50 µg*Patent Pending.

Japan +81 (0)3 5225 3638

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3

DNA Demethylation

NEW: 5-Hydroxymethylcytosine – The Newest Epigenetic Mark, and theFirst Antibody for Its Study
DNA is packaged through its incorporation into chromatin, the wrapping of DNA around histone proteins, whichfacilitates higher-order organization of the genome. This packing of DNA into chromatin has many heritable effects
upon DNA-dependent processes. ‘Epigenetic’ changes like histone modiﬁcations exist in addition to epigenetic
events that occur on the DNA itself. CpG dinucleotide clusters, or ‘islands’, scattered throughout the genome (upto 50% of human gene promoters contain CpG islands) are subject to methylation of the 5th carbon position of thecytosine. DNA methylation participates in genome-wide processes such as the regulation of gene expression, genomic
imprinting, silencing of repetitive elements and, when aberrant, contributes to cancer. DNA methylation can also
recruit methyl-CpG binding proteins (MeCP2 and the MBD family) that are effectors of downstream regulatory events.
The enzymes that catalyze the addition of the methyl group to DNA are
well characterized, but the process bywhich CpG methylation is reversed is
not straightforward. It has recently been
described that a series of enzymatic
reactions leading to the deamination of
5-methylcytosine (5-mC) results in theremoval of the methylated base, as thedeaminated 5-mC leads to a DNA
mismatch that is repaired by the
nucleotide excision system.
In 2009, a second potential pathway
for DNA demethylation was identiﬁed,
coincident with the discovery of an alternative form of DNA CpG methylation.
Kriaucionis and Heintz reported on therelative abundance of 5-hydroxymethylcytosine (5-hmC) in a speciﬁc cell type inthe brain in which the genome is organized to a large extent into euchromatin.
Another group identiﬁed the enzymatic
pathway by which 5-mC is converted
to 5-hmC (Tahiliani et al.) by the TET1
enzyme and related proteins (Figure 1).
5-hmC is replaced with cytosine by DNA
repair proteins, effectively leading todemethylation of the CpG dinucleotide.
References

Huang et al. (2010) PLoS One 26: e8888.
Jin et al. (2010) Nucl Acids Res in press.
Kriaucionis and Heintz (2009) Science 324: 924-929.
Nestor et al. (2010) Biotechniques 48: 317-319
Tahiliani et al. (2009) Science 324: 930-935.

4

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The identiﬁcation of this novel DNA
modiﬁcation requires that new tools becreated in order to study the role of thisevent. None of the traditional methodsof DNA methylation analysis apply to
5-hmC. Bisulﬁte treatment of 5-hmC
turns it into cytosine 5-methylenesulfonate (CMS), which makes it appear
to be 5-mC in downstream analysis.
Also, the typical enzymatic (methylation sensitive restriction enzymes) andenrichment (by antibody or methylbinding protein) techniques don’t workon 5-hmC.
New tool enables the study of 5-hmC
To facilitate the study of this new andpotentially important regulatory event,
Active Motif has developed the ﬁrst
antibody that recognizes 5-hmC, our
5-Hydroxymethylcytidine antibody. It
speciﬁcally recognizes DNA that contains
5-hmC, but it does not recognize DNA
containing 5-mC (Figure 2).

Figure 1: Conversion of 5-mC to 5-hmC.
The methyl group at position 5 of 5-methylcytosine isoxidized to 5-hydroxymethylcytosine by the TET familyof iron-dependent oxygenases.

Europe +32 (0)2 653 0001

Figure 2: Dot blots using 5-hmC and 5-mC antibodies.
Top panel: 5-Hydroxymethylcytidine antibody(Catalog No. 39769; 1:10,000 dilution).
Bottom panel: 5-Methylcytidine antibody(1:1,000 dilution).
Lane 1: DNA derived from mouse embryonic stem
cells (150 ng).
Lane 2: DNA derived from mouse spleen (600 ng).
Lane 3: A 27-base oligonucleotide containing
5-Hydroxymethylcytosine (1.2 ng).
Lane 4: A 24-base oligonucleotide containing
5-Methylcytosine (2000 ng).

Why do you need Active Motif’s new
5-hmC antibody?
• It discriminates 5-hmC from 5-mC;
only 5-hmC is recognized• Enrichment by existing 5-mC
antibodies doesn’t work on 5-hmC• Enzymatic approaches and bisulﬁtesequencing cannot differentiate
5-mC from 5-hmC in DNA• Methyl-CpG DNA binding proteins
do not recognize 5-hmC
To ﬁnd out more about Active Motif’s
ground-breaking new 5-Hydroxymethylcytidine antibody, please visit us atwww.activemotif.com/5hmc.

Japan +81 (0)3 5225 3638

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May 2010 • volume 11 • number 2

DNA Methylation & Enrichment Assays

NEW: Improved Format for Methylated CpG Island Enrichment
Due to the scientiﬁc importance of CpG shores and regions of low-density methylation, the MethylCollector™ Ultra
Kit* has been modiﬁed to include both low and high salt binding buffers. This improved format provides a better
enrichment solution for efficient capture of low-density CpG methylation.
MethylCollector™ Ultra advantages• Detection from 1 ng – 1 µg DNA• Works on double-stranded DNA
fragmented by sonication or
enzymatic digestion• MBD2b/MBD3L1 heterodimeric
protein capture is more efficientthan MeDIP or MBD2 alone• Proteinase K elution step eliminates
the need for time consuming salt
gradient elution protocols• Results in less than 3 hours• Enriched DNA is suitable for use inendpoint or real time PCR, WGA,
microarray analysis or sequencingA?`^_jXck)''YgLeYfle[<clk\[

CfnjXck<clk\[

LeYfle[@eglk(''YgB?`^_jXck<clk\[<clk\[,''YgLeYfle[LeYfle[CfnjXck<clk\[<clk\[

LeYfle[LeYfle[

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Figure 1: MethylCollector Ultra speciﬁcally enriches for CpG-methylated DNA.
MethylCollector Ultra was performed with 100 ng Mse I digested human, male genomic DNA using both high and low
salt binding conditions. Eluted DNA was cleaned and analyzed in endpoint PCR for 32 cycles. A) The methylated NBR2
promoter, which contains 7 CpGs, is specifically enriched and eluted under both binding conditions. B) The unmethylated
APC promoter, which contains 29 CpGs, remains in the unbound fraction for both binding conditions, illustrating thespecificity of the MBD2b/MBD3L1 heterodimeric protein at enriching only DNA with methylated CpG dinucleotides.

Speciﬁcally Detect Unmethylated CpG Islands for Better Validation of Results
Active Motif’s UnMethylCollector™ Kit** is the ﬁrst commercially available kit for the specific isolation andenrichment of unmethylated CpG dinucleotides. Instead of relying on negative results from methyl-speciﬁc
binding techniques to identify hypomethylated promoters, UnMethylCollector offers a speciﬁc, reliable
technique that provides a positive readout, which is a much better method to obtain or validate your results.
How does UnMethylCollector™ work?
UnMethylCollector uses a His-tagged
CXXC domain to speciﬁcally bind theunmethylated CpG islands of genomic
DNA fragments, which have been
prepared by sonication or enzymatic
digestion. The protein-DNA complexes
are captured with nickel-coated magnetic beads. Subsequent wash steps are
performed using an optimized buffer
and the included magnet to separate
unmethylated fragments from the restof the genomic DNA. The enriched,unmethylated CpG islands are theneluted in the presence of high salt.

Even more methylation research tools
For bisulﬁte conversion analysis, don’t
forget to check out Active Motif’s
MethylDetector™ Kit. And, our 5-Methylcytidine monoclonal antibody is greatfor MeDIP assays. For complete productlistings and additional details on all of
the DNA methylation assays and antibodies offered by Active Motif, please
visit www.activemotif.com/dnamt.

Product

FormatCatalog No.

MethylCollector™ Ultra

30 rxns

55005

UnMethylCollector™

30 rxns

55004

MethylDetector™

50 rxns

55001

Fully Methylated Jurkat DNA

10 µg

55003

Jurkat genomic DNA

10 µg

55007

5-Methylcytidine monoclonal antibody

50 µg

39649

Get 15% off UnMethylCollector™ when you buy it with MethylCollector™ Ultra.
Visit www.activemotif.com/promo for complete details.

**Patent Pending.
* MethylCollector Ultra & MethylCollector are covered under U.S. Patent No. 7,425,415.

www.activemotif.com

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North America 877 222 9543

5

Magnetic bead-based Chromatin IP

Choose the ChIP Kit That is Best for Your Intended Application,
From the Source that Developed Them All
The different ways in which ChIP is used have multiplied in recent years, making it critical to correctly match thespeciﬁc kit with the intended application. Active Motif has a long history of innovation in kits for different ChIP
applications, having simpliﬁed the protocol with magnetic beads, developed enzymatic shearing procedures,
introduced a high-throughput ChIP kit, and a Re-ChIP kit for sequential ChIP assays on a single sample. Let us
help you make ChIP a simple and productive endeavor.
The New Leader in ChIP
Active Motif was ﬁrst to introduce ChIP
kits employing magnetic beads, and wehave only increased our efforts since.
We offer ChIP-IT™ Express HT, whichenables you to perform 96 ChIPs simultaneously, and Re-ChIP-IT™, which solves
the complexities of performing sequential ChIP assays on a single sample; weoffer your choice of enzymatic digestionor sonication for chromatin shearing,as well as the most complete line of
ChIP-validated chromatin antibodies,
which we design and produce ourselvesin conjunction with our ChIP kits (seewww.activemotif.com/chipabs). Withothers, ChIP is an interesting afterthought. For us, ChIP is our core identity.
Advantages of magnetic beads
The ChIP-IT Express Kit is our ﬂagship
ChIP product, which was created andreﬁned by years of work at Active Motif.
The result is a 1-day ChIP protocol that

Figure 1: PCR of chromatin IP performed on 100,000 cells.
Typically, ChIP requires 2 million cells per reaction. With
ChIP-IT Express, it is possible to perform successful ChIP
with 100,000 cells or less. In the gel above, ChIP was
performed in duplicate on chromatin isolated from
100,000 HeLa cells using the Negative Control IgG, RNA
pol II antibody and GAPDH PCR primers supplied in the
ChIP-IT Control Kit – Human (Cat. No. 53010).

6

North America 877 222 9543

Figure 2: True high-throughput ChIP.
With the efficient plate-based protocol of ChIP-IT Express
HT, you can process up to 96 ChIP reactions at a time.

utilizes magnetic beads, which provide
a number of advantages over traditional
ChIP methods:• No pre-blocking needed –magnetic beads are inert• Compatible with ChIP-Sequencing –no salmon-sperm DNA is used in the
protocol• No need for DNA purification –our specialized ChIP Elution Buffer
eliminates the need for expensive
DNA puriﬁcation columns• Perform ChIP with fewer cells –routinely perform chromatin IP with
100,000 cells or fewer (Figure 1)• Optimized components, buffers& protocols – Active Motif sweats
all the details, so you don’t have toWhich ChIP-IT Kit is right foryour application?
Table 1 (on page 7) gives a brief overview
of the different features of our various
ChIP-IT Express and ChIP-IT Kits.

ChIP-IT Express HT: High-throughput
ChIP is now a reality
Performing multiple ChIP experiments
simultaneously using traditional methods
only compounds the difﬁculty of theassay. That is why Active Motif developed ChIP-IT Express HT as the solution
to this problem. Now, it is possible toperform the time-saving ChIP-IT Express
protocol in a 96-well format (Figure 2),
with everything optimized to the level
you have come to expect from Active
Motif. ChIP-IT Express HT is compatible
with our enzymatic or sonication-based
shearing kits for chromatin preparation,
as well as with our ChIP-IT Control Kits.
Re-ChIP-IT for Sequential ChIP
experiments from one sample
Deciphering the Histone Code often
requires showing that two marks or
associated proteins occur at the samesite in the genome. Sequential ChIP (orRe-ChIP) involves sequential chromatin
immunoprecipitations using different
antibodies in series on the same sample.
Sequential chromatin IP was technically challenging and difﬁcult, until now.
Active Motif’s Re-ChIP-IT Kit has been
optimized for this technique, making it
easy to perform sequential ChIP, so you
can more easily localize two different
proteins or histone modiﬁcations to thesame genetic locus (Figure 3, next page).

Get 30% off up to 2 antibodies when you buy them with any ChIP-IT Kit.
Visit www.activemotif.com/promo for complete details.
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May 2010 • volume 11 • number 2

Shearing Method
Sonication

Magnetic

IP Method

What next? If your DNA sample isscarce, amplify it
If you started with a low cell number,chances are the chromatin recovered
after ChIP is limiting, constraining theexperiments you can do next. To help,
Active Motif offers its GenoMatrix™
Whole Genome Ampliﬁcation Kit,
which makes it possible to amplify all
recovered DNA fragments. The kit uses
a new approach that virtually eliminates
representational bias, so the ampliﬁed
DNA pool contains the same sequence
representation as the starting material(Figure 4). For more information, please
visit www.activemotif.com/wga.

Magnetic bead-based Chromatin IP

3530

Ct Value25
20

Enzymatic

ChIP-IT™ Express Enzymatic
ChIP-IT™ Express• Includes protein G• Includes protein G
magnetic beads, whichmagnetic beads, which
reduce background, take
reduce background, takeless time and enable theless time and enable theuse of less material
use of less material
Shearing: Enzymatic
Shearing: Sonication
Controls: Offered separately Controls: Offered separately

ChIP-IT™
• Includes protein G
agarose beads for standard centrifugation inCentrifugation
the IP and wash steps
Shearing: Sonication
Controls: Included (human)

ChIP-IT™ Enzymatic• Includes protein G
agarose beads for standard centrifugation in
the IP and wash steps
Shearing: EnzymaticControls: Included (human)

Table 1: Brief overview of the various shearing and IP/washing options offered in the ChIP-IT Express and ChIP-IT Kits.

15
10
5
0

BRCA1

BRCA2

CREB1

DHFR

FosB

MAS1

NEDD4

NFκB

Figure 4: Representative and quantitative DNA
ampliﬁcation without sequence bias.
Quantitative real-time PCR was carried out on DNA
samples using primers indicated below each data set
and the Ct value for each sample plotted. The DNA wasderived from an original DNA sample amplified using the
GenoMatrix Whole Genome Amplification Kit (red bars),
amplified with a competitor’s whole genome amplification kit (copper bars) or not subjected to whole genome
amplification (purple bars).

Choose from the widest variety of ChIP
kits and accessories available
Active Motif is committed to offering comprehensive solutions for ChIP,
enabling you to do your experimentswith the conﬁdence that our products
have been optimized and improved overmany years. In addition to our kits, weoffer useful reagents like Ready-to-ChIP
Chromatin and species-speciﬁc ChIP-IT
Control Kits. Let our experience help you
work through the challenges of ChIP,
which will free up your time and energy
for designing your experiments. For
more complete information, please give
us a call or visit us on the web atwww.activemotif.com/chip.

www.activemotif.com

Figure 3: Sequential chromatin immunoprecipitation using Re-ChIP-IT.
The lane numbers are the same in each panel to indicate that the DNA is from the same chromatin sample. The left panel
shows the results of PCR performed on an aliquot of DNA removed from the experiment after the first ChIP step; the rightpanel shows PCR results on DNA from chromatin samples after both ChIP steps. For example, chromatin samples subjected
to first ChIP using Mouse IgG as a negative control (lanes 1 and 2 in the left panel) were then subjected to a second ChIP
with an RNA Pol II antibody (lanes 1 and 2 in the right panel). Chromatin samples in which Mouse IgG was used as either the
first antibody (lanes 1 and 2) or second antibody (lanes 5 and 6) show little amplification of GAPDH DNA in either the left
(first ChIP) or right panel (first and second ChIP). Chromatin samples in which the first antibody used was anti-RNA Pol II
and the second antibody was anti-TFIIB (lanes 3 and 4) show good amplification of GAPDH DNA after the second ChIP
(right panel) indicating co-localization of RNA Pol II and TFIIB at the same region of the GAPDH promoter.

Product

FormatCatalog No.

ChIP-IT™ Express

25 rxns

53008

ChIP-IT™ Express Enzymatic

25 rxns

53009

ChIP-IT™ Express HT

96 rxns

53018

Re-ChIP-IT™

25 rxns

53016

GenoMatrix™ Whole Genome Ampliﬁcation Kit

50 rxns

58001

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North America 877 222 9543

7

Phospho-Protein & Transcription Factor Detection

Simple, Effective Monitoring of Phosphorylation Using In-cell Detection
Active Motif’s Fast Activated Cell-based ELISA (FACE™) Kits are a simple,
sensitive, cell-based method to detect protein phosphorylation directly inthe cell, without the trouble of making cell extracts or running gels.

In-cell ELISAs: Cell Signaling made easy
Active Motif’s In-cell ELISAs require
just 2 hours of hands-on time. Cells aregrown in 96-well culture plates, stimulated with an agonist, ﬁxed in formaldehyde, and probed separately with anantibody to the protein in question anda phospho-speciﬁc antibody to a siteon the same protein. Detection is with asecondary antibody and your choice ofcolorimetric or chemiluminescent readout. Comparing the two signals readily
gives you a relative phosphorylation
readout (Figure 1), and your results are

2

3

4
43 kDa1

easily normalized to cell number using
the provided Crystal Violet Dye.

2

3

A variety of kits to choose from
FACE Kits are available for over 20 different targets (see web address below).
The Suspension Cell FACE module wasdesigned to work with all FACE Kits; it
improves results when working with suspension cells by providing 96-well ﬁlter
plates that make it easier to performwashing & liquid handling steps. And,
with FACE Maker Kits, you can use yourown primary and secondary antibodies
to detect any target or modiﬁcation
state of interest.

A

4
43 kDaOD450 nmFACE advantages• Cell based – it stays on the plate• Accurate – combination of two
antibody readouts reliably gives
relative phosphorylation state• Fast – 5 hour protocol

1

B

Phospho p38
Total p38

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

C

0

0.1
1
10
Anisomycin induction (µg/ml)

Figure 1: Phospho and total p38 MAPK assays.
Macrophage 4/4 cells were grown in 10 cm dishes to
80% confluency, serum-starved for 16 hours and stimulated with anisomycin for 15 minutes. Cell lysates weremade and Western blots performed using phospho- (A)and total-p38 antibodies (B). For FACE, 4/4 cells weregrown in 96-well plates, stimulated as above, fixed andthen assayed in triplicate using the FACE p38 Kit (C). Data
were corrected for cell number through use of the kit’s
Crystal Violet Dye. Western blot data provided courtesyof Dr. Henri H. Versteeg and Dr. Maikel P. Peppelenbosch.

Simplify your phospho-assays today!
For complete information on the FACE
product line, please give us a call or visit
us at www.activemotif.com/face.

Detect 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 activity. TransAM Family Kits enable you to measure the activity of entire families oftranscription factors in a single experiment. In addition to being the most cited transcription factor DNA-binding
assay in literature references, Active Motif also 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.

8

North America 877 222 9543

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.

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

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May 2010 • volume 11 • number 2

Polycomb & ChIP-validated Antibodies

Antibodies for the Study of Polycomb Group Proteins
The organization of DNA into chromatin has broad effects on genome based processes, especially gene expression
and chromosome & chromatin structure. Open chromatin structure is a hallmark of transcriptionally active regionsof the genome (euchromatin), whereas silenced, inactive regions of the genome are found in a more compact
chromatin architecture (heterochromatin). Some regions of the genome that contain developmentally regulated
genes can switch from one state to the other (facultative heterochromatin).
The Polycomb group proteins areimportant players in regulating facultative heterochromatin and the expression
state of genes found there. In doing
so, they help regulate cell fate andlineage decisions. Polycomb proteinsare found primarily in two complexes:
PRC2, which is recruited to Polycomb
regulated regions through interactions
with DNA binding proteins, and PRC1,
involved in the spreading and maintenance of the repressed state. For example, PRC2 contains the EZH2 histone
methyltransferase, speciﬁc for lysine 27

of histone H3. Polycomb group proteinsfunction in the maintenance of cellular
identity in hematopoietic stem cells,
embryonic stem cells and many different
tissue types in vertebrate development.
Additionally, they play a role in cancer,
as EZH2 levels are elevated in prostatecancer (see page 11).
Figure 1: Ring1B staining in U2OS cells.
Human USOS osteosarcoma cells were stained with
Ring1B monoclonal antibody (Catalog No. 39663) andvisualized by indirect immunofluorescence.

Active Motif offers a variety of wellcharacterized antibodies to assist in the
study of Polycomb proteins. For more
information, please visit us atwww.activemotif.com/polycombabs.

ChIP-validated Histone Monoclonal Antibodies Ensure Your Success
Eliminate the lot-to-lot variability often found with polyclonals by using
Active Motif’s ChIP-validated histone modiﬁcation monoclonal antibodies.
Manufacturing a ChIP-validated
polyclonal antibody to a histone or
histone modiﬁcation is time consuming
and labor intensive. And, once the ﬁrst
batch has been used up, you must startover again from scratch, with no guarantee of success, or even that the second
batch of antibody will perform similarly
to the ﬁrst. These potentiallyinconsistent batch-to-batch process
issues can cause problems when
conducting studies that require a
consistent methodological approach,such that all your data are comparable. When performing a techniqueas painstaking and tricky as chromatin
immunoprecipitation, consistency isparamount.

www.activemotif.com

Figure 1. Chromatin IP using Histone H3 dimethyl Lys9
monoclonal antibody (Clone MABI 0307).
Chromatin IP was performed using the ChIP-IT Express
Kit (Catalog No. 53008) and 100 µl of Ready-to-ChIP
HeLa Chromatin (Catalog No. 53015) per ChIP. DNA wasthen purified from the immunoprecipitated chromatin
and a region of the human HOXD13 promoter was
amplified by PCR. Lane 1: ChIP using 10 µg of Histone
H3 dimethyl Lys9 mAb. Lane 2: PCR input control.
Lane 3: ChIP using negative control mouse IgG.

To eliminate the batch-to-batch inconsistencies of polyclonals, Active Motif
offers a range of ChIP-validated monoclonals. So, you can be sure you are getting the same antibody every time, nomatter when you need it. For more, visit
www.activemotif.com/hismodmono.
Product

FormatCatalog No.

Histone H2B ubiquityl Lys120 antibody (Clone 56)

100 µg39623

Histone H3 antibody (Clone MABI 0301)

100 µg39763

Histone H3 monomethyl Lys4 antibody (Clone MABI 0302)

100 µg39635

Histone H3 dimethyl Lys4 antibody (Clone MABI 0303)

100 µg39679

Histone H3 monomethyl Lys9 antibody (Clone MABI 0306)

100 µg39681

Histone H3 dimethyl Lys9 antibody (Clone MABI 0307)

100 µg39683

Histone H3 acetyl Lys27 antibody (Clone MABI 0309)

100 µg39685

Histone H3 trimethyl Lys27 antibody

200 µl

39535

Histone H4 trimethyl Lys20 antibody (Clone MABI 6F8-D9)

100 µg

3967

Japan +81 (0)3 5225 3638

Europe +32 (0)2 653 0001

North America 877 222 9543

9

Histone Modification ELISAs

NEW: Screen Histone PTMs with a Fast, Sensitive ELISA
Post-translational modiﬁcations (PTMs) on histone tails have been shown to correlate very closely with the nuclear
processes of transcription, chromosome packaging and DNA damage repair. Due to the importance of these posttranslational modiﬁcations, Active Motif has expanded its line of Histone Modiﬁcation ELISAs. Kits are now available
for the detection of lysine methylation at K4, K9 and K27, or serine phosphorylation at S10 and S28 on histone H3.

Each Histone Modiﬁcation ELISA is also
tested against other modiﬁcations toensure speciﬁcity (Figure 2). Validated
modiﬁcation-speciﬁc controls areincluded in each kit.

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How do the Histone ELISAs work?
The Histone Modiﬁcation ELISA Kits
provide a simple, sensitive method for
detecting changes in speciﬁc histone
modiﬁcations, such as methylation or
phosphorylation, from puriﬁed core
histones or histones isolated by acid extraction. These kits are sandwich ELISAs
that utilize a capture antibody against
histone H3 and a detecting antibody
speciﬁc to the modiﬁcation of interest.
An HRP-conjugated secondary antibody
and developing solutions provide asensitive colorimetric readout (Figure 1).

(%)

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(

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'%/
'%'%+
'%)
'

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(,%-

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Figure 2: Histone H3 monomethyl Lys27 speciﬁcity.
Recombinant Histone H3, mono-, di- and trimethyl Lys27
proteins were assayed from 15 ng - 1 µg per well using
the Histone H3 monomethyl Lys27 ELISA. The results
show low background with other modifications,demonstrating the specificity of the assay.

Histone Modiﬁcation ELISA advantages• Sensitive – works with puriﬁed core
histones or acid extracted samples• Fast – assay can be completed inless than 3 hours• Flexible – 96-stripwell plate allows
screening in low or high throughput• Controls ensure success – modiﬁcation speciﬁc controls included• Colorimetric assay – easily quantiﬁed by spectrophotometry on a
microplate reader at 450 nm

Validated controls also available
For further analysis of histone posttranslational modiﬁcations, site- anddegree-speciﬁc methylated proteins thatare provided for use as a reference standard curve in each histone methylation
ELISA are also available separately (page3). Each recombinant histone is veriﬁedby high-resolution mass spectrometry,
dot blot and immunoblot to conﬁrm
the methylation reaction is over 99%
complete and the recombinant histoneis speciﬁc for the target of interest.
For researchers looking to study epigenetic events using acid extracts, Active
Motif offers untreated and treated HeLa
acid extracts, such as the paclitaxel
treated HeLa acid extracts included in
the histone phosphorylation ELISAs.
To learn more about our recombinanthistones, acid extracts and associatedhistone modiﬁcation antibodies, please
visit us at www.activemotif.com.

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(''

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Gli`]`\[ ?\CX
:fi\
8Z`[
?`jkfe\j <okiXZkFigure 1: Histone H3 trimethyl Lys4 sensitivity.
The Histone H3 trimethyl Lys4 ELISA was used to assay
10 µg HeLa core histones purified using Active Motif’s
Histone Purification Mini Kit (Catalog No. 40026) and10 µg HeLa acid extract. The Recombinant Histone H3
trimethyl Lys4 protein provided in the kit was assayedfrom 1.5-100 ng/well as a reference standard curve.

For more information about our Histone
Modiﬁcation ELISAs, please call or visit
www.activemotif.com/hiselisa.

10North America 877 222 9543

Product

FormatCatalog No.

Histone H3 monomethyl Lys4 ELISA NEW

1 x 96 rxns

53101

Histone H3 dimethyl Lys4 ELISA NEW

1 x 96 rxns

53112

Histone H3 trimethyl Lys4 ELISA NEW

1 x 96 rxns

53113

Histone H3 dimethyl Lys9 ELISA

1 x 96 rxns

53108

Histone H3 trimethyl Lys9 ELISA

1 x 96 rxns

53109

Histone H3 monomethyl Lys27 ELISA NEW

1 x 96 rxns

53104

Histone H3 trimethyl Lys27 ELISA

1 x 96 rxns

53106

Histone H3 phospho Ser10 ELISA

1 x 96 rxns

53111

Histone H3 phospho Ser28 ELISA

1 x 96 rxns

53100

Total Histone H3 ELISA

1 x 96 rxns

53110

Histone Puriﬁcation Mini Kit

20 rxns

40026

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com

May 2010 • volume 11 • number 2

Histones and Histone Modifications

Using Histone and Histone Modiﬁcations as Biomarkers for Cancer
In eukaryotes, DNA is organized into chromatin through the wrapping of DNA around histone octamers to form thenucleosome. These histone proteins are subject to a variety of post-translational modiﬁcations (phosphorylation,acetylation, methylation…) that are involved in genome-dependent processes like transcription, chromosome
organization and the like. These modiﬁcations, while important regulators of chromatin-mediated processes,
can also be indicators of diseases such as cancer.
Recently, researchers at the University ofCalifornia at Los Angeles identiﬁed particular histone modiﬁcations as potential
biomarkers for the clinical outcome ofpancreatic adenocarcinoma. By examining tissue samples from patients withpancreatic cancer, it was determinedthat low levels of either histone H3lysine 4 dimethyl (H3K4me2), histone H3lysine 9 dimethyl (H3K9me2), or histone
H3 lysine 18 acetyl (H3K18ac) were associated with poor survival. Low levelsof H3K4me2 and H3K18ac combined
were the most signiﬁcant indicator ofclinical outcome. Says senior researcher
Dr. David Dawson, “Using a speciallydevised test and algorithm, we were able
to discriminate two groups of pancreaticcancer patients who were more or lesslikely to have longer disease-freeremissions and overall survival.”
Indeed, many examples of connections
between histone modiﬁcations andcancer exist already (Table 1). Speciﬁc
patterns of histone levels or histone
modiﬁcations have been correlatedwith particular cancers. Additionally,
increased expression of the histone H3lysine 27 methyltransferase EZH2 isobserved in prostate cancer. It is possible,
and likely, that soon many more histone
modiﬁcations will be identiﬁed as
markers for other types of cancer.
Histone research simpliﬁed
To facilitate your research into histone
modiﬁcations and their role in diseases
like cancer, Active Motif has a variety ofhigh-quality tools at your disposal.

www.activemotif.com

DiseaseConnection

Reference

Breast cancer progression Histone H2A.Z levels

Hua et al. (2008) Mol Syst Biol. 4: 188.
Svotelis et al. (2010) Cell Cycle 9: 364-370.

Chronic Lymphocytic
Leukemia

Histone H2A levels

Su et al. (2009) Proteomics 9 :1197-1206.

Lung cancer recurrence

Histone macroH2A levels

Sporn et al. (2009) Oncogene 28: 3423-3428.

Non small cell lung cancer Histone acetylation and methylation

Barlesi et al. (2007) J. Clinic. Oncol. 25: 4358-4364.

Pancreatic cancer

H3 acetylation and methylation

Manuyakorn et al. (2010) J. Clin. Oncol. 28: 1358-1365.*

Prostate cancer

H3 and H4 acetylation and methylation Seligson et al. (2005).

Prostate cancer

Histone methyltransferase EZH2

Varambally et al. (2002).

Skin and other cancers

H4 acetylation and methylation

Fraga et al. (2005) Nature Genet. 37: 391-400.

Squamous cell carcinoma

H3 and H4 acetylation and methylation I et al. (2010) Cancer Epidem Biomark Prev.
19:566-573.

*Data in this paper were generated in part using Active Motif’s Histone H3 acetyl Lys18 pAb (Catalog No. 39693).

Table 1: Connections that have been shown between histone modiﬁcations and cancer types.

We offer a variety of antibodies tohistones and histone modiﬁcations
validated in applications like ChIP
(page 9), Westerns and IF. Active Motifis the only company to offer histone
puriﬁcation kits that enrich histones
out of human cells and PBMCs. We alsooffer a broad range of ELISAs to measurelevels of histone modiﬁcations from acidextracts or puriﬁed histones (page 10).
And, we offer a ﬂuorescence polarization binding assay that enables you
to identify proteins that interact withhistone H3 methylated at either Lys9
or Lys27 (Figure 1). Please visit the links
below to learn more.
Product Line

Figure 1: Binding of HP1 to histone H3 Methyl-Lys9.
One µM of fluorescent histone H3 Lys 9 peptides, which
are either unmodified or mono-, di- or trimethylated
at K9, were added to a 96-well plate containing a serial
dilution of HP1 protein (x axis) starting at a concentration of 200 µM. The plate was scanned on a Tecan
Infinite F200 using 485 nm excitation and 535 nm
emission filters with polarizers. A gain of 80 was used;
100 reads were taken of each well, then averaged.

Web Address

Histone Modification FP Binding Assays

www.activemotif.com/hilite

Histone Modification ELISAs

www.activemotif.com/hiselisa

Histone Purification Kits

www.activemotif.com/histonepur

Histone and Histone Modification Antibodies

www.activemotif.com/histoneabs

Recombinant Histone Modifying Enzymes

www.activemotif.com/hismodenz

Recombinant Modified Histone Proteinswww.activemotif.com/recombhis

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North America 877 222 9543

11