IN THIS ISSUE

Transcription, DNA Methylation & Histone Modiﬁcations

NEW

Epigenetics
Research Services(see page 9)

2 Epigenetics: Inﬂuencing the Kinetics of
NFκB Signal Transduction

7 Screen for Histone Interactions Using theMODiﬁed™ Histone Peptide Array

3 Non-radioactive Methods to Evaluate
Transcription Factor Binding

8 NEW: Obtain Analysis-ready DNA with the
Chromatin IP DNA Puriﬁcation Kit

4 NEW: 5-Hydroxymethylcytosine Antibodies
Enhance DNA Methylation Studies

9 NEW: Active Motif Now Offers Genome-wide
Analysis Services for Epigenetics

5 NEW: hMeDIP Assay Simpliﬁes the
Study of 5-hmC DNA Methylation

9 NEW: 5-Hydroxymethylcytosine MeDIP-Seq
and MeDIP-chip Services

6 Histone Puriﬁcation Kits that Preserve
Post-translational Modiﬁcations

10 Study Transcriptional Regulation with
ChIP-validated Transcription Factor Abs

6 NEW: Recombinant Histone Proteins toAnalyze Site-speciﬁc Modiﬁcations

10 Explore Chromatin Biology and Epigenetics
using Antibodies Validated for use in
Immunoﬂuorescence

7 NEW: Histone ELISAs for Sensitive
Detection of Acetylated Histones

11 Reagents for High-quality Fluorescence
Microscopy

NFκB Activation & Signaling

Epigenetics: Inﬂuencing the Kinetics of NFκB Signal Transduction
The nuclear factor-κB (NFκB)
signaling pathway is widely studied
due to its involvement in theregulation of genes that control
inﬂammation, immune response,
cell survival, cell proliferation andhuman disease.
The NFκB Rel family of transcription factors consists of a set of
evolutionarily conserved DNAbinding proteins that includes p65,
p50, p52, RelB and c-Rel. Inactive
NFκB is sequestered in the cytoplasm by the IκB family of inhibitory proteins. Activation of the
NFκB signaling pathway induces
phosphorylation of IκB proteinsby IκB kinases (IKK). Depending on
the type of stimulation, the classical (also referred to as ‘canonical’)or the ‘non-canonical’ NFκB signaling pathways can be activated(Figure 1). Phosphorylation by the
IKK complex leads to ubiquitination and degradation of theinhibitor IκB molecules by theproteasome. The liberated NFκB
complexes translocate to the
nucleus where they bind DNA andregulate gene expression by binding to κB DNA-binding sites.

2

North America 877 222 9543

Figure 1: The NFκB signaling pathway.
Depiction of the NFκB canonical and non-canonical signaling pathways.

Evidence is building that epigenetic modiﬁcations play a critical rolein orchestrating NFκB-mediatedtranscriptional activation. Thereis a signiﬁcant understanding of
the contribution of the synergistic
effects of various cofactors andsignaling networks on the spatiotemporal coordination of NFκBmediated transcriptional eventsat the cellular level. However, theﬁne-tuning and kinetics of the response to those signals at the genome level can best be explainedby analyzing how histone modiﬁcation patterns inﬂuence and are, inturn, inﬂuenced by the activity of

NFκB. In particular, recent studies
have focused on how epigenetic
events inﬂuence the accessibilityof the NFκB transcription factor/
cofactor enhanceosome complex
to target κB DNA-binding sites
within the chromatin structure.
Active Motif offers a variety of
products to analyze the complex
dynamics of NFκB transcription
factor binding and associatedepigenetic events. To learn more
about Active Motif’s NFκB-related
products, please give us a callor visit us on our website atwww.activemotif.com/nfkb.

Save 10% on products for NFκB research.
For a limited time, get 10% off select products for NFκB.
Just cite NFKB11 when you order. For complete details and a list ofeligible products, please visit www.activemotif.com/promo.
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NFκB Activation & Signaling

March 2011 • volume 12 • number 1

Non-radioactive Methods to Evaluate Transcription Factor Binding

Active Motif offers the Gelshift™ Chemiluminescent EMSA Kit as a simple,
convenient, non-radioactive solution for assessing variant transcription factor
binding to DNA targets. The non-radioactive format displays enhanced sensitivity when compared to traditional 32P or digoxigenin EMSA methods.
Product

FormatCatalog No.

TransAM™ NFκB Family (p50, p52, p65, c-Rel & RelB)

2 x 96 rxns

43296

TransAM™ NFκB p50

1 x 96 rxns

41096

TransAM™ NFκB p52

1 x 96 rxns

48196

TransAM™ NFκB p65

1 x 96 rxns

40096

Gelshift™ Chemiluminescent EMSA

100 rxns

37341

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To facilitate the study of DNA-binding activity, Active Motif offers its TransAM™ Kits, which are highly-sensitive,
non-radioactive transcription factor ELISAs that enable detection of even the smallest changes in the levels ofactivated transcription factors. In addition, the 96-stripwell format allows for both high and low throughput screening of samples. Its sensitivity, ﬂexibility and speciﬁcity make the TransAM method the most published alternative
to electrophoretic mobility shift assays (EMSA). To view a complete list of available TransAM™ Kits, please visit us atwww.activemotif.com/transam. Recombinant proteins are also available foruse as standard curves in the TransAM NFκB p50 and TransAM NFκB p65 Kits.
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and radioactive gelshift.
Activated NFκB p50/DNA-binding results are shown forhuman fibroblast WI-38 cells that were stimulated with
IL-1α for 30 minutes. Increasing amounts of whole-cell
extracts were assayed using either TransAM NFκB p50(A) or gelshift (B). The TransAM method is 10-fold moresensitive and provides quantitative data.

Additional Tools to Aid your NFκB Research (for Custom Services, see page 9)
Phospho-protein Detection• FACE™ NFκB p65 Proﬁler (Ser468 & Ser536)• FunctionELISA IκBαLuciferase Reporter Assays• RapidReporter® pRR-High-NFκB vector• RapidReporter® pRR-High-NFκB Assay• RapidReporter® Gaussia Luciferase Assay
In Vivo Fluorescent Labeling• LigandLink™ pLL-1-NFκB p65
SUMOylation• SUMOlink™ SUMO-1• SUMOlink™ SUMO-2/3

Antibodies• Highly characterized antibodies including: IκBα, IKKα,IKKβ, IKKγ, NFκB p50, NFκB p65, NFκB p100 and more
www.activemotif.com/abs

Extracts• Variety of untreated and stimulated nuclear, cytoplasmic
and whole-cell extracts to study NFκB signaling
www.activemotif.com/extracts

Recombinant Proteins• NFκB-related recombinant proteins, including NFκB p50,
NFκB p65, IKKβ and others
www.activemotif.com/proteins

To learn more about all of Active Motif’s NFκB-related products, please visit www.activemotif.com/nfkb.

www.activemotif.com

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3

5-Hydroxymethylcytidine Abs & Standards

NEW: 5-Hydroxymethylcytosine Antibodies Enhance DNA Methylation Studies
Conventional methods for studying DNA methylation (enrichment by antibody or methylated-DNA binding protein,
methylation sensitive enzyme digestion and bisulﬁte sequencing) cannot distinguish 5-hydroxymethylcytosine (5-hmC)
from 5-methylcytosine (5-mC). To help study this novel form of DNA methylation, Active Motif offers two
polyclonal antibodies and a new monoclonal antibody that are speciﬁc for the 5-hmC modiﬁcation. Like all of
our antibodies for epigenetics research, they are highly speciﬁc and have been validated for use in various practicalapplications, including methylated DNA immunoprecipitation (MeDIP), immunoﬂuorescence and dot blot.
The importance of DNA methylation
DNA methylation is an epigenetic
event in which DNA methyltransferases(DNMTs) catalyze the reaction of amethyl group to the ﬁfth carbon ofcytosine in a CpG dinucleotide. This
modiﬁcation helps to regulate gene expression and is also involved in genomic
imprinting, while aberrant DNA methylation is often associated with disease.
5-methylcytosine (5-mC) is a type of
DNA methylation found in plants andvertebrates. A second type of DNA
methylation exists, 5-hydroxymethylcytosine (5-hmC), which results from the
enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by
the TET family of cytosine oxygenases(Figure 1). Elevated levels of 5-hydroxymethylcytosine have been observed inneurons and embryonic stem cells. It is
possible that 5-hmC represents a pathway by which DNA is demethylated, as
5-hydroxymethylcytosine is recognizedby the DNA mismatch repair system andreplaced with unmethylated cytosine.

Figure 1: Schematic of conversion of 5-mC to 5-hmC.
5-methylcytosine is converted to 5-hydroxymethylcytosine by the TET family of enzymes.

4

North America 877 222 9543

Figure 2: 5-Hydroxymethylcytidine monoclonal
antibody tested by dot blot analysis.
DNA samples were spotted (indicated in ng on the left)
and blotted with 5-Hydroxymethylcytidine monoclonal
antibody (Clone 59.1) at a dilution of 0.2 µg/ml.
Lanes 1-3: double-stranded DNA.
Lanes 4-6: single-stranded DNA.
Lanes 1 & 4: DNA containing 5-hydroxymethylcytosine.
Lanes 2 & 5: DNA containing 5-methylcytosine.
Lanes 3 & 6: unmethylated DNA.

Methylated DNA Standard Kit
In addition to the antibodies to study
DNA methylation, Active Motif offers
our Methylated DNA Standard Kit. This
kit includes three recombinant DNA
standards derived from the APC gene
regulatory region: unmethylated DNA,
5-methylcytosine methylated DNA and
5-hydroxymethylcytosine methylatedDNA. This kit (which also includes PCRprimers speciﬁc to the APC gene) can beused to provide controls for experiments
studying the different types of DNA
methylation (see Figure 1 on page 5).

Antibodies to study DNA methylation
Active Motif has three antibodies speciﬁc for the 5-hydroxymethylcytosine
form of DNA methylation. Our newest
release, the 5-Hydroxymethylcytidine
mouse monoclonal antibody (Clone 59.1)
has been validated for use in MeDIP and
dot blot (Figure 2). Like our polyclonal
antibodies, the new monoclonal does
not require DNA to be denatured toits single-stranded form in order towork in a MeDIP experiment. This canbe advantageous when analyzing theresulting DNA using high-throughput
Next-Gen sequencing. We also offer
the more traditional 5-Methylcytidine
mouse monoclonal antibody to study
5-mC DNA methylation in a variety of
applications.
Full line of DNA Methylation products
Active Motif offers a variety of antibodies, assay kits and services for theepigenetics and chromatin researchcommunity. To learn more about the different DNA methylation tools available,
visit www.activemotif.com/dnamt. For
a complete listing of DNA methylation
antibodies, including application data,
visit www.activemotif.com/dnamethabs.

Product

FormatCatalog No.

5-Hydroxymethylcytidine mAb (Clone 59.1)

100 µg39999

5-Hydroxymethylcytidine antibody (rabbit IgG)

100 µg39791

5-Hydroxymethylcytidine antibody (rabbit serum)

100 µl39769

5-Methylcytidine antibody mAb (Clone 33D3)

50 µg

39649

Methylated DNA Standard Kit

3 x 2.5 µg

55008Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com

March 2011 • volume 12 • number 1

MeDIP & hMeDIP Assays

NEW: hMeDIP Assay Simpliﬁes the Study of 5-hmC DNA Methylation
Active Motif’s new hMeDIP Kit helps simplify the analysis of 5-hydroxymethylcytosine methylation. Methylated DNA
Immunoprecipitation (MeDIP) is an immunocapture technique in which an antibody speciﬁc for methylated cytosines
is used to immunoprecipitate methylated genomic DNA fragments. The hMeDIP Assay uses a highly speciﬁc, puriﬁed
5-hydroxymethylcytidine (5-hmC) antibody to selectively enrich for DNA fragments with 5-hydroxymethylcytosine
methylation from the rest of the genomic DNA population. Some advantages of the hMeDIP Kit are that it not only
differentiates between 5-hydroxymethylcytosine DNA and 5-methylcytosine DNA, but also that the fragmented
genomic DNA used in the assay can be double-stranded DNA, which prevents the problems associated with linker
bias when preparing the enriched DNA for downstream Next-Gen sequencing.

Additionally, the hMeDIP technique isable to enrich for DNA fragments containing cytosine methylation regardlessof the sequence context. While most
DNA methylation in mammalian tissues
occurs in a CpG context, studies havefound that 15-20% of total cytosine
methylation in embryonic stem (ES) cells
occurs at sequences other than CpG.
These features make the hMeDIP Kit
ideal for researchers interested in identiﬁcation of total cytosine methylation,or in differentiating between 5-mC and5-hmC methylation.

Kit

Antibody

DNA Input

Input Range

hMeDIP

5-hmC puriﬁed rabbit pAb

double-stranded or
single-stranded DNA

100 ng – 1 µg

Table 1: hMeDIP Kit sample requirements.

Included methylated DNA controls
The hMeDIP Kit also includes methylated
DNA controls for use in determining the
efﬁciency of the enrichment. The controls are 338 base pair DNA fragmentsthat are completely unmethylated,
5-methylcytosine methylated or
5-hydroxymethylcytosine methylated.
When these control DNAs are spiked
into sample DNA the speciﬁcity of thecapture antibody can be conﬁrmed via
real time PCR with the included PCRprimers (Figure 1). The Methylated DNA
Standards used as controls in thehMeDIP Kit can also be purchased
separately (see page 4).
Visit www.activemotif.com/dnamt tolearn more about the wide variety of
DNA methylation analysis tools andassay kits offered by Active Motif.

Product

FormathMeDIP

10 rxns

55010

MeDIP COMING SOON

10 rxns

55009

www.activemotif.com

Japan +81 (0)3 5225 3638

Catalog No.

Europe +32 (0)2 653 0001

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Why use hMeDIP over other DNA
methylation enrichment techniques?
Unlike traditional methods used to study
DNA methylation, such as enzymatic
approaches & bisulﬁte conversion thatcannot directly differentiate between
5-mC and 5-hmC DNA methylation, or
methyl CpG binding protein enrichment
which can only recognize and bind 5-mC
methylation, hMeDIP is able to use theselective afﬁnity of the immunocapture antibody to distinguish between
5-methylcytosine and 5-hydroxymethylcytosine DNA methylation.

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^c[:D7hWXX_j?]=Figure 1: hMeDIP results using “spiked” control DNA.
Mse I digested human genomic DNA (500 ng) was spiked
with 25 pg of either methylated (meDNA), hydroxymethylated (hmeDNA) or unmethylated (unDNA) DNA
controls. These samples were processed using thehMeDIP Kit with the 5-hmC antibody and compared to anegative control rabbit IgG antibody. Real time PCR with
the included APC PCR primers was run and the recovered DNA was plotted as a percentage of the total input
DNA (500 ng). The results show the hMeDIP Kit is specific for enrichment of 5-hydroxymethylcytosine DNA.

As part of its recent acquisitionof Genpathway, Active Motif now
offers a 5-Hydroxymethylcytosine
MeDIP-Seq service. For details,
please see page 9 or visit
www.genpathway.com.

North America 877 222 9543

5

Analysis of Histone Post-translational Modifications

Complete Analysis of Histone Post-translational Modiﬁcations
Whether you are studying transcription factors such as NFκB or chromatin-modifying proteins, understanding theimplications of histone post-translational modiﬁcations as they relate to gene regulation and chromatin context willbe critical to determining how these modiﬁcations affect or contribute to disease. Active Motif’s unique portfolioof histone technologies provides researchers with a complete solution for analysis. To either download or request a
copy of our Histone Analysis Products brochure by mail, please visit www.activemotif.com/info.

Histone Puriﬁcation Kits that Preserve Post-translational Modiﬁcations
Histone Puriﬁcation Kit advantages
Active Motif’s unique Histone Puriﬁcation and Histone Puriﬁcation Mini Kits
enable the isolation of core histones
from any cell culture or tissue sample
while preserving their post-translational
modiﬁcations (Figure 1). Unlike standard
acid extraction techniques, it is possible
to isolate core histones as either a single
fraction or to further separate them into
H2A/H2B and H3/H4 fractions.
Which kit is right for you?
• Histone Puriﬁcation – can processsamples of up to 2.5 mg which canbe eluted as either a single fraction
with all four histones or as separate
H2A/H2B and H3/H4 fractions• Histone Puriﬁcation Mini – has acapacity of up to 0.5 mg per sample
which is eluted as a single fraction
containing H2A, H2B, H3 and H4

Acetylation

Methylation

Phosphorylation

Figure 1: Active Motif’s Histone Puriﬁcation Kits preserve post-translational modiﬁcations.
Acetylation, methylation and phosphorylation states are preserved as well or better with the Histone Purification Kit thanstandard acid extraction methods.

Product

FormatCatalog No.

Price ($US)

Histone Puriﬁcation Kit

10 rxns

40025

275

Histone Puriﬁcation Mini Kit

20 rxns

40026

275

NEW: Recombinant Histone Proteins to Analyze Site-speciﬁc Modiﬁcations
Recombinant histone proteins withsite-speciﬁc modiﬁcations
Active Motif continues to expand its
line of recombinant methylated andacetylated histones. We have added
16 new recombinant proteins to ourportfolio, including methylation of lysine
14, 18 and 23 on histone H3 and methylation of lysine 5 and 16 on histone H4.
Additionally, we now offer recombinanthistone H3 acetylated at lysine 23.

6

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Why use recombinant histones?
These recombinant proteins moreclosely mimic “natural” histones than
peptides, making them ideal substrates
for functional assays and activity
screens. The recombinant proteins are
suitable for use with Active Motif’s
histone modifying enzymes and are even
included in our Histone Modiﬁcation
ELISAs to generate standard curves forhistone quantiﬁcation.

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New biotinylated recombinant H3
To expand the versatility of our recombinant histones, Active Motif now offers
Recombinant Histone H3 biotinylated at
the N-terminus.
To see an up-to-date list of the morethan 40 recombinant histone proteins
currently available, please visit us atwww.activemotif.com/recombhis.

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March 2011 • volume 12 • number 1

Analysis of Histone Post-translational Modifications

NEW: Histone ELISAs for Sensitive Detection of Acetylated Histones
Quantify your histone modiﬁcations
The Histone Modiﬁcation ELISA Kits
offer a quick and easy way to screen andquantify histone modiﬁcations withinyour sample. The kits are designed as
sandwich ELISAs that utilize a histone
H3 capture antibody and a detecting
antibody that is speciﬁc for the modiﬁed
residue of interest.

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Active Motif currently offers morethan ten different Histone ELISAs formethylation and phosphorylation marks
on histone H3. Now, Active Motif has
expanded its line of ELISAs to include
assays to detect acetylation of histone
H3 lysine 9 and lysine 14. Each kit also
includes a positive control recombinant
protein that can be used to generate astandard curve for histone quantiﬁcation(Figure 1). The Total Histone H3 ELISA isavailable to measure total histone levels
or to normalize quantities of modiﬁed
histones from the sample.

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Figure 1: Detection of histone H3 lysine 9 acetylation in puriﬁed core histones and acid extracted histones.
The Histone H3 acetyl Lys9 ELISA was used to quantitate the amount of histone H3 lysine 9 acetylation present in HeLa
core histones made using Active Motif’s Histone Purification Mini Kit (Cat. No. 40026, page 6) and HeLa acid extracts. The
Recombinant Histone H3 acetyl Lys 9 protein that is provided in the kit was assayed from 0.039 - 2.5 ng/well as a standard
curve. Data was quantified against the linear range of the standard curve. Data shown are from wells assayed in duplicate.

Product

FormatCatalog No.

Histone H3 acetyl Lys9 ELISA

1 x 96 rxns

53114

Histone H3 acetyl Lys14 ELISA

1 x 96 rxns

53115

Total Histone H3 ELISA

1 x 96 rxns

53110

To see a complete list of the over 10 Histone Modiﬁcation ELISAs available, including
methylation and phosphorylation ELISAs, please visit www.activemotif.com/hiselisa.

Screen for Histone Interactions Using the MODiﬁed™ Histone Peptide Array
Unique array to screen interactions
The MODiﬁed™ Histone Peptide Arrayenables analysis of antibody, protein andenzyme interactions with histones andtheir post-translational modiﬁcations.
The array contains 384 different combinations of acetylation, phosphorylation,
methylation and citrullination modiﬁcations on the N-terminal tails of histones
H2A, H2B, H3 and H4.
To learn more about the MODiﬁed
Histone Peptide array, or to download
the free Array Analyse software, please
visit www.activemotif.com/modiﬁed.

MODiﬁed Array advantages• Histone speciﬁc – unique arraypanel tests for speciﬁc histone
modiﬁcations• Study neighboring effects – each
peptide contains up to four modiﬁcation combinations, enabling theanalysis of the effects of neighboring modiﬁcations• Detects like a Western blot – fastand easy to use; works with either
ECL-based or colorimetric detection• Free software for analysis – measures spot intensity and generates
Excel-compatible ﬁles for analysis

Product

FormatCatalog No.

MODiﬁed™ Histone Peptide Array

1 array
5 arrays

13001
13005

MODiﬁed™ Array Labeling Kit

5 rxns13006

www.activemotif.com

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7

8

9

Figure 1: 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.

North America 877 222 9543

7

Chromatin IP DNA Purification

NEW: Obtain Analysis-ready DNA with the Chromatin IP DNA Puriﬁcation Kit
Chromatin Immunoprecipitation (ChIP) is a powerful, well-established technique for studying interactions between
chromatin-associated proteins and speciﬁc regions of the genome. While the use of ChIP in combination withgenome-wide analysis techniques can yield a tremendous amount of information regarding the distribution oftranscription factors and histone modiﬁcations, most downstream analysis techniques require DNA that has been
puriﬁed away from the components and contaminants present in your eluted ChIP sample. Active Motif’s Chromatin
IP DNA Puriﬁcation Kit enables you to quickly and easily clean up your ChIP DNA samples to make them ready for
analysis, eliminating the need for labor intensive and time consuming phenol/chloroform extraction.
What is the Chromatin IP DNA
Puriﬁcation Kit process?
Once your ChIP experiments are complete, the DNA puriﬁcation procedurecan be started immediately. The entire
procedure takes only ﬁve to ten minutes,
depending upon the number of samples
to purify. The Chromatin IP DNA Puriﬁcation Kit is compatible with samples
from all of Active Motif ChIP-IT™ Kits,
or from any standard chromatin IP kit orprocedure, whether they use mechanical or enzymatic shearing of chromatin,
agarose or paramagnetic bead enrichment. It can also be used to purify DNA
from other Active Motif kits, including
the new hMeDIP Kit, MethylCollector™
Ultra and UnMethylCollector™. The new
Chromatin IP DNA Puriﬁcation Kit isdesigned for use with a microcentrifuge
for sample processing, but can also be
used with a vacuum manifold.
Because binding of DNA to the silica
matrix of the DNA puriﬁcation column
is pH-dependant, the kit’s Binding Buffer
contains a convenient pH indicator dye.
This makes it possible for you to see
the pH of your samples before applying them to the column, helping ensure
successful puriﬁcation of your ChIP DNA(Figure 1). After binding, the DNA on thecolumn is washed, then eluted using theincluded Elution Buffer.

8

North America 877 222 9543

DNA recovery and yield
After elution of the DNA, you now
have samples of high quality and purity,
suitable for a number of downstream
analysis techniques, including PCR (endpoint or quantitative), Southern blotting,microarray analysis or Next-Gen sequencing (Figure 2). Depending upon thenumber of cell equivalents of chromatinused in each ChIP reaction, the amountof recovered DNA will be from 100 ng to
1 µg. DNA can be successfully recoveredfrom ChIP experiments starting withas few as 10,000 cells. DNA fragments
below 50 base pairs in length are not recovered efﬁciently, but this shouldn’t be
a problem as the recommended average
size for chromatin fragments in a typical
ChIP reaction is 500 base pairs.

Figure 1: DNA Binding Buffer pH indicator dye.
The DNA Purification Binding Buffer has a pH indicator
dye so the pH of the solution can be easily determined.
The DNA should only be applied to the column if thesolution is bright yellow (left), indicating a pH under 7.5.
DNA will not bind to the column if the pH is higher than
7.5. (NaOAc is included to reduce sample pH, if needed.)

Figure 2: Quantitative PCR performed on ChIP DNA.
Quantitative PCR was performed using DNA purified
with the Chromatin IP DNA Purification Kit after ChIP
using the indicated antibodies (ChIP Abs). Chromatin
IP experiments were performed using the ChIP-IT
Express Kit (Catalog No. 53008) and Ready-to-ChIP HeLa
Chromatin (Catalog No. 53015, 7.5 x 105 cell equivalents
per ChIP). Quantitative PCR was carried out using primers specific for the indicated gene (Locus). The normalized data represent fold enrichment over ChIP experiments carried out with control IgG.

Chromatin IP DNA Puriﬁcation Kit
advantages• Optimized for use with epigenetics
applications• Compatible with all of Active Motif’s
ChIP-IT Kits, as well as with kitsfrom other manufacturers• Purify DNA ChIP’d using agarose orparamagnetic bead methods• Convenient pH indicator to ensure
proper binding of your samples• Can also be used to purify DNA
from our hMeDIP, MethylCollectorand UnMethylCollector Kits
To ﬁnd out more about our new Chromatin IP DNA Puriﬁcation Kit, please visit
us at www.activemotif.com/dnapure.

Product

FormatCatalog No.

Chromatin IP DNA Puriﬁcation Kit

50 rxns

58002

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com

March 2011 • volume 12 • number 1

Epigenetic Services

NEW: Active Motif Now Offers Genome-wide Analysis Services for Epigenetics
As part of its recent acquisition of Genpathway, genome-wide analysis services are now offered by Active Motif.
This is the perfect combination of products and know-how. The leader in kits and reagents for the study ofchromatin biology and epigenetics now offers services and expertise to facilitate your research into transcription
factor distribution and function, histone modiﬁcations and DNA methylation.
• FactorPath™ – discovery, identiﬁcation and quantitation of transcription factor and cofactor bindingsites across the genome• TranscriptionPath™ – discovery andidentiﬁcation of actively transcribedgenes at the DNA level to detect
genome-wide changes in gene
expression within minutes oftreatment• MethylPath™ – discovery, identiﬁcation and quantitation of methylated
DNA regions; also includes our new
5-hmC MeDIP-Seq and bisulﬁtesequencing services• HistonePath™ – map histone
modiﬁcations and/or enzymes
that regulate histone modiﬁcations
across the genome• Bisulﬁte Sequencing – determine
the locations and methylation
status of cytosines?*B).d\*<Q?)^\e\j"jkiXe[^\e\j$jkiXe[

Figure 1: ChIP-Seq data for histone H3 lysine 27 methylation and EZH2.
Chromatin IP was performed using antibodies specific for H3K27me3 and EZH2, the H3K27 methyltransferase, followed byhigh-throughput sequencing of the ChIP DNA. The data are presented in an 8 million base pair window and shows nearly
identical genomic localization for histone H3 lysine 27 methylation and the enzyme responsible for depositing the mark.
Shown is a region of chromosome 10q surrounding the HPSE2 gene.

In addition to the services listed to the left, we also offer customized assay development and speciﬁc services to optimize ChIP-based procedures, including antibody qualiﬁcation and data analysis. To learn more about our genome-wide analysis services forchromatin biology, transcription, histones and DNA methylation, please visit our website
at www.activemotif.com/services or email us at services@activemotif.com.

NEW: 5-Hydroxymethylcytosine MeDIP-Seq and MeDIP-chip Services
Due to the importance of DNA methylation in biology and disease, Active Motif
now offers 5-hydroxymethylcytosine
MeDIP-Seq and MeDIP-chip as custom
services. Just provide us with frozen
biological samples or puriﬁed DNA and
we will perform the hMeDIP-Seq or
hMeDIP-chip experiments, then provide
you with, and help you interpret, the data.
Our hMeDIP genome-wide analysis services allow you to study 5-hmC methylation on a genome-wide scale utilizing
Active Motif’s expertise and researchtools without having to be an expert in
the techniques yourself. The advantage
Active Motif has over other companies

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is that our proprietary monoclonal
antibody recognizing 5-hydroxymethylcytosine methylation does not requirethat the DNA be made single-stranded

before immunoprecipitation. The ability
to use double-stranded DNA eliminates
linker bias in the downstream processing
steps prior to Next-Gen sequencing.
<c\mXk\[j`^eXc`e^\e\Yf[`\j^\e\j"jkiXe[
Figure 1: 5-hMeDIP-chip performed on human brain DNA.
Human brain DNA (2 µg) was immunoprecipitated with 10 µg of 5-Hydroxymethylcytidine antibody (Clone 59.1, Catalog No.
39999). Following hMeDIP, the DNA was amplified, labeled and hybridized to an Affymetrix Human Tiling 2.0R Array. Shown
is a region from chromosome 6q containing the ARID1B, ZDHHC14 and SNX9 genes. The results show that 5-hydroxymethylcytosine is enriched primarily in the coding region of genes, rather than the promoter or regulatory regions.

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9

ChIP- & Immunofluorescence-validated Antibodies

Study Transcriptional Regulation with ChIP-validated Transcription Factor Abs
Gene expression depends on the recognition of speciﬁc promoter sequencesby transcriptional regulatory proteins,which in turn recruit other proteins, such
as RNA polymerase, in order to effecttranscription. In the human genome,
there are at least 2,600 genes that encode proteins involved in transcriptional
regulation. These fall into three maincategories: sequence speciﬁc DNA binding transcription factors (proteins that
directly bind speciﬁc DNA regulatory
sequences), general transcription factors(those associated with RNA polymerase
holoenzyme complex) and transcriptional co-regulators (proteins recruitedby the DNA binding factors).
Because one of the most important
techniques used to study the function
and genomic localization of these pro-teins is chromatin immunoprecipitation(ChIP), Active Motif offers an extensive
line of ChIP-validated antibodies toimportant regulatory factors (Figure 1).

As part of its acquisition of
Genpathway, Active Motif now
offers FactorPath™ genome-wide
transcription factor binding
identiﬁcation and analysis as acustom service. See page 9 formore details, or visit us atwww.activemotif.com/factorpath.
To ﬁnd out more about our ChIPvalidated antibodies to transcriptional
regulators, please call or visit us atwww.activemotif.com/tfchipabs.

Figure 1: Quantitative PCR performed on RNA pol II
CTD phospho Ser5 antibody ChIP’d DNA
Chromatin IP was performed using the ChIP-IT™ Express
Kit (Catalog No. 53008) and HeLa chromatin (1.5 x 106 cell
equivalents per ChIP) with 10 µg RNA pol II CTD phospho
Ser5 antibody (Catalog No. 39749) or the equivalentamount of rabbit IgG as a negative control. Real time,quantitative PCR (RT-qPCR) was performed on DNA purified from each of the ChIP reactions using a primer pair
specific for the PABPC1 gene. Data are presented as foldenrichment of the ChIP antibody signal versus the negative control IgG (arbitrarily assigned a value of 1) using
the ddCT method.

Explore Chromatin Biology and Epigenetics using Antibodies Validated for use
in Immunoﬂuorescence
Visualizing a protein in its native cellularenvironment using immunoﬂuorescencemicroscopy requires the use of very
high-quality antibodies. They must be
speciﬁc, of high titer and able to recognize native (non-denatured) protein. To
assist you in your study of chromatin
biology, epigenetics and transcription,
Active Motif offers antibodies validated
for use in immunoﬂuorescence to a wide
range of protein targets including:• Chromatin-modifying proteins• Histones and histone modiﬁcations• Transcription factors• Nuclear receptors• Regulators of cell structure andfunction

10

North America 877 222 9543

Figure 1: HDAC2 visualized by immunoﬂuorescence.
HeLa cells were stained with HDAC2 monoclonal antibody (Clone 3F3, Catalog No. 39533) at a 1:1000 dilution.
Red: HDAC2 mAb (Clone 3F3) Green: alpha-Tubulin
mouse monoclonal (Clone 5-B-1-2, Catalog No. 39527)
conjugated to Chromeo™ 488.

Figure 2: Aurora B visualized by immunoﬂuorescence.
HeLa cells were stained with Aurora B polyclonal
antibody (Catalog No. 39261) at a dilution of 1:200 andalpha-Tubulin monoclonal antibody (Clone 5-B-1-2,
Catalog No. 39527) used at a dilution of 1:500.
Red: Aurora B. Green: alpha-Tubulin. Blue: DAPI.

Active Motif offers a wide range ofprimary antibodies validated for use inimmunoﬂuorescence. For more information, visit www.activemotif.com/ifabs.

To ﬁnd out more about our high-quality
ﬂuorescent secondary antibodies, visit
www.activemotif.com/secondary.

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March 2011 • volume 12 • number 1

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 novel high-resolution ﬂuorescence microscopy (e.g. STimulated Emission Depletion, or STED). We use thesereagents in our daily work to develop novel assays and to evaluate our antibodies in a consistent and reliable manner.
Fluorescent secondary antibodies
Active Motif offers goat anti-mouseand goat anti-rabbit secondary antibodies that are conjugated to a number ofhigh-quality dyes, including Chromeo™
ﬂuorescent dyes (Table 1). Our optimized
conjugation method, coupled withsubsequent puriﬁcation of the conju-

gate, makes our ﬂuorescent secondaries brighter than other commerciallyavailable conjugates and lowers the
background in many applications. Active Motif’s antibody conjugates havebeen tested in applications such as ﬂow
cytometry and wideﬁeld, confocal andhigh-resolution ﬂuorescence microscopy.

Fluorescent Dye

Absorption

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*

Table 1: Fluorescent properties of dyes when conjugated to secondary antibodies.
* Alexa Fluor dyes are a registered trademark of Life Technologies™.

MAX Stain™ Immunoﬂuorescence Tools
MAX Stain Immunoﬂuorescence Tools
eliminate the challenge of getting
consistent high-quality ﬂuorescentimages by providing a complete set of
optimized reagents. 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
and MAXwash reagents.

• MAXblock™ Blocking Medium –effectively blocks non-speciﬁc
antibody binding without reducing
signal intensity or speciﬁcity• MAXbind™ Staining Medium –increases the antibody binding
speciﬁcity and intensity of yourIF 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

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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.

Why use Chromeo™ conjugatedsecondary antibodies?
• Brightness – high ﬂuorescentintensity improves sensitivity• Limited photobleaching – enables
multiple exposures and increased
exposure times• Flexibility – conjugates work undermultiple ﬁxation conditions• Speciﬁcity – low ﬂuorescentbackground
Fluorescent Chromeo™ Dyes
In addition to the secondary antibody
conjugates, Chromeo™ Dyes are alsoavailable as NHS-Esters, Carboxylic Acids,
Azides and Alkynes for Click-Chemistry.
Biotin and Streptavidin conjugates arealso available. To see a complete list ofavailable ﬂuorescent dyes, please visit us
at www.activemotif.com/dyes.

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11

Visit us at www.activemotif.com

Active Motif develops validated antibodies
and assays that enable the discovery andcharacterization of key epigenetic processes.
Our products are developed in-house andsupported by scientists with expertise inchromatin biology. For a complete productlisting please visit www.activemotif.com.
CHROMATIN ANALYSISChIP kits and ChIP-validated antibodies
HISTONE MODIFICATION
Antibodies & ELISAs, Arrays, HAT/HDAC assays,
Histone puriﬁcation and Recombinant Histones
DNA METHYLATION
Methylated DNA enrichment, bisulﬁte conversion,
DNMT assays, whole genome ampliﬁcation,
hMeDIP, 5-hmC & 5-mC antibodies

Superior Antibodies & Kits for

EPIGENETICS RESEARCH