IN THIS ISSUE

Antibodies, Kits and Services for Epigenetic Research
2 NEW: Antibodies to 5-Carboxylcystosine &5-Formylcytosine Modiﬁcations

8 HAT & HDAC Assays to Easily Screen
for Changes in Histone Acetylation

2 NEW: DNA Standard to Study
5-Carboxylcytosine

8 Antibodies & Enzymes for the
Study of Histone Acetylation

3 Tools to Analyze All Aspects of
DNA Methylation

9 Navigate the Active Motif Website
with the Greatest of Ease

4 Avoid Antibody Bias with Covalent Labelingof 5-Hydroxymethylcytosine

10 NEW: Antibodies and Reagents forSuper-resolution GSDIM Microscopy

5 NEW: MeDIP-Seq Now Offered by
Active Motif Epigenetic Services

11 NEW: ChIP-IT® Control Kits forReal Time PCR Analysis

6 Polycomb Proteins Help Determine
Embryonic Stem Cell Identity

11 Expansive Offering of Species-validated
Control qPCR Primer Sets for ChIP

5-Carboxylcytosine & 5-formylcytosine DNA Methylation

NEW: Antibodies to 5-Carboxylcystosine & 5-Formylcytosine Modiﬁcations
Active Motif is proud to be at the forefront of research and discovery with the introduction of our new DNA
modiﬁcation antibodies for 5-carboxylcytosine (5-caC) and 5-formylcytosine (5-fC) modiﬁcations. While recent
studies have focused on the ability of the TET family of cytosine oxygenase enzymes to convert 5-methylcytosine
into 5-hydroxymethylcytosine, it now appears that further oxidation of 5-hydroxymethylcytosine results in the formation of 5-formylcytosine and 5-carboxylcytosine.
Even more DNA modiﬁcations?
Two recent publications in Science by Ito
et al. and He et al., demonstrated thatTet proteins can oxidize 5-hydroxymethylcytosine to generate 5-formylcytosine
and 5-carboxylcytosine. Ito et al. went
on to show that 5-fC and 5-caC arepresent in mouse embryonic stem cells,
while 5-fC could also be identiﬁed in
major mouse organs. Following the oxidative pathway where 5-methylcytosine
is converted to 5-carboxylcytosine (seepage 3), it is then believed that 5-caC is
excised from genomic DNA by thymine
DNA glycosylase (TDG); thereby illustrating a mechanism of DNA demethylation.
Further analysis of 5-fC and 5-caC by
Inoue et al. in Cell Research, revealed
that 5-fC and 5-caC appear in thepaternal pronucleus after fertilization,
concomitant with the disappearance of
5-methylcytosine, and that the levels of
5-fC and 5-caC are gradually diluted out
by DNA replication (Figure 1).
Working with the lab of Yi Zhang, Howard Hughes Medical Institute Investigator at the University of North Carolina
at Chapel Hill, Active Motif now offers
antibodies against these cytosine derivatives. The antibodies have been characterized in dot blot and immunostaining
experiments and are available as either
whole-rabbit serum or puriﬁed IgG. Visit
www.activemotif.com/dnamethabs for acomplete list of available antibodies.
References

Ito et al. (2011) Science 333: 1300-1303.
He et al. (2011) Science 333: 1303-1307.
Inoue et al. (2011) Cell Research 21: 1670-1676.

2

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5-fC
5-mC
DAPI

5-caC
5-mC
DAPI

Figure 1: Replication-dependent loss of 5-fC and 5-caC revealed in a 2-cell metaphase embryo.
Shown are representative immunofluorescent images of mitotic chromosome spreads that have been co-stained with
Active Motif’s 5-Formylcytosine (5-fC) or 5-Carboxylcytosine (5-caC) antibodies (red, Catalog Nos. 61223 and 61225,
respectively), a 5-methylcytosine (5-mC) antibody (green) and DAPI (blue) at the two-cell stage of mouse preimplantation
development. The 5-fC and 5-caC antibodies were used at a 1:2000 dilution. The images reveal that at the two-cell stage,
only one of the two sister chromatids is enriched for 5-fC and 5-caC, consistent with findings that 5-fC and 5-caC levels
are diminished by half in blastomeres with each round of DNA replication (Inoue et al. (2011)).

NEW: DNA Standard to Study 5-Carboxylcytosine
Control DNA for positive conﬁrmation
To complement the antibodies now
available for 5-carboxylcytosine, Active
Motif also offers the 5-Carboxylcytosine
DNA Standard. This double-stranded
DNA sequence contains a total of
12 carboxylcytosine modiﬁcations,(8 modiﬁcations on the forward strand
and 4 modiﬁcations on the reverse
strand). The 5-Carboxylcytosine DNA
Standard contains both modiﬁed andunmodiﬁed DNA standards with enoughmaterial to perform ten dot blots each.

Figure 2: 5-Carboxylcytosine DNA standard.
Varying amounts of dsDNA from the Methylated DNA
Standard Kit (Cat No. 55008) or 5-Carboxylcytosine DNA
Standard were spotted onto a nylon membrane andprobed with Active Motif’s 5-Carboxylcytosine antibody(Catalog No. 61225, 1:2000 dilution).

Product

FormatCatalog No.

5-Carboxylcytosine DNA Standard

0.5 µg55014

5-Carboxylcytosine pAb (Serum)

100 µl61225

5-Carboxylcytosine pAb (IgG)

100 µg61229

5-Formylcytosine pAb (Serum)

100 µl61223

5-Formylcytosine pAb (IgG)

100 µg61227

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March 2012 • volume 13 • number 1

DNA Methylation

Tools to Analyze All Aspects of DNA Methylation
The identiﬁcation of 5-hydroxymethylcytosine (5-hmC), a derivative of the well-characterized 5-methylcytosine(5-mC) epigenetic mark, established a precedent for the role of Tet enzymes in catalyzing DNA demethylation as amechanism for epigenetic reprogramming. Recent studies reveal that the pathway of demethylation may involve
further oxidation of 5-hmC to 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) during cell differentiation anddevelopment. To aid researchers in furthering their understanding of how DNA methylation and demethylation
work in concert to regulate gene expression, development, and stem cell identity, Active Motif has developed an
expansive line of products and technologies to address the most current and cutting edge areas in DNA methylation
research. Let our antibodies, assays and enzymes deliver the results you need.

The Active Motif advantage• Convenience – one-stop shop for
all your DNA methylation products• Choice – expansive offering ofproducts including antibodies, kits
and recombinant proteins• Cutting edge – products andtechnologies relevant to the mostcurrent research• Consistency – our optimized
reagents guarantee consistent andreliable results

www.activemotif.com• CGBP Antibody• Methylated DNA Standard Kit

Decarboxylase

DNMT• DNMT Activity/
Inhibition Assay• DNMT Antibodies• HP1 Antibodies• MBD Antibodies• MeCP2 Antibodies• Uhrf1 Antibody

Tet• MethylCollector™ Ultra• UnMethylCollector™
• MeDIP• MethylCollector™
• 5-mC Antibodies• Methylated DNA
Standard Kit• Recombinant Tet1 Protein• Hydroxymethyl Collector™
• hMeDIP• PvuRts1I• β-Glucosyltransferase• 5-hmC Antibodies• Methylated DNA
Standard Kit• 5-caC Antibody• 5-Carboxylcytosine DNA Standard

Tet

One-stop shop for all your DNA
methylation products
To ensure that you have the necessary
tools you need at your disposal tosucceed with your DNA methylation
research, Active Motif offers an expansive portfolio of products to cover thefull spectrum of DNA methylation analysis (see Figure 1). Whether your studies
require the use of antibodies to distinguish between methyl, hydroxymethyl,
formyl, and carboxyl cytosine modiﬁcations, Tet enzymes to use for 5-hmC
conversion assays, or kits to enrich formethylated or hydroxymethylated DNA,
Active Motif has you covered witha wide range of antibodies, kits, andrecombinant proteins to address yourspeciﬁc research needs. For complete
ordering information or to learn more
about all of Active Motif’s DNA methylation products, please call or visit us atwww.activemotif.com/dnamt.

Tet• Recombinant
Tet1 Protein• Recombinant Tet1 Protein• 5-fC Antibody

Figure 1: Schematic representation of the oxidation of 5-methylcytosine by Tet enzymes and related products.
Tet enzymes catalyze the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine(5-fC), and 5-carboxylcytosine (5-caC) via oxidation of the methyl group at the 5-position on the cytosine ring. The schematic
depicts the demethylation pathway and the Active Motif products associated with the individual steps of the reaction.

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3

5-Hydroxymethylcytosine Enrichment

Avoid Antibody Bias with Covalent Labeling of 5-Hydroxymethylcytosine
Active Motif’s Hydroxymethyl Collector™ Kit is the only commercially available technology to utilize a covalent labeling method to enrich for DNA fragments containing 5-hydroxymethylcytosine (5-hmC). The covalent labeling method
avoids sequence and density bias that can be seen with antibody enrichment methods. This method also enables
greater sensitivity in the detection of 5-hmC fragments and allows for more complete DNA methylation analysis.

The modiﬁed 5-hmC DNA is thencovalently linked to a biotin conjugate.
Magnetic streptavidin beads and theincluded bar magnet are used to capture
the biotinylated 5-hmC DNA fragments. Due to the high afﬁnity between
biotin and streptavidin, Hydroxymethyl
Collector is capable of capturing DNA
fragments with as few as two hydroxymethylcytosine residues per DNA strand.
The biotin/streptavidin interaction also
allows for high stringency washing conditions to minimize non-speciﬁc binding.
Elution buffer cleaves the linker between the biotin and the glucosyl-5hydroxymethylcytosine, releasing theenriched DNA from the magnetic beads.
The eluted DNA is collected and puriﬁed
with the included puriﬁcation reagents,
leaving you with DNA that is readyfor analysis of individual loci by PCR(Figure 2), or whole-genome analysis by
microarray or sequencing.
References

1. Song et al. (2010) Nature Biotechnology 29: 68-72.
2. Szwagierczak et al. (2010) Nucl. Acids Res., 38: e181.

4

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OH

Biotin

NH2

N3
N

N
H

HO
HO

N3

O
NH2

O
OH

β-Glucosyltransferase+ UDP-Azide-Glucose(1 hr at 37°C)

HO
HO

2. Modified
Glucosyl-5-hmC

O

N
H

Biotin Conjugate
Solution(1 hr at 37°C)

SA

Enriched DNA
containing
5-hmC

N3
HO
HO

O

NH2

OH

Elute the DNA
from the Biotin(30 min at RT)N

5. Eluted DNA is purified using
the included reagents.

N
H

HO
HO

O

N
O

N
H

Magnetic
Streptavidin Beads(30 min at RT)
N3

O

NH2

3. Biotinylated
Glucosyl-5-hmC

Biotin

O

O

OH

N

O

1. Fragmented DNA
containing 5-hmC

O
O

NH2

OH

N

4. DNA is captured forwashing and elution.

N
HO

Figure 1: Flow chart of Hydroxymethyl Collector method.
Fragmented DNA (100-500 bp size range) is modified by addition of a glucose moiety. A biotin conjugate is then covalently
attached to the glucose enabling capture with magnetic streptavidin beads. Enriched DNA is eluted by cleavage of the biotin linker to release the DNA from the magnetic beads. Eluted DNA is purified before use in downstream applications.
>oZhenoc[j^ob9ebb[Yjeh()('e\?dfkj

How does it work?
The Hydroxymethyl Collector™ method
utilizes the ability of β-glucosyltransferase to modify 5-hydroxymethylcytosine residues by addition of a glucose
moiety. When fragmented genomic DNA
is incubated in the presence of β-glucosyltransferase and a UDP-Azide-Glucose
donor, the enzyme modiﬁes the 5-hmC
residue, creating glucosyl-5-hydroxymethylcytosine1. Modiﬁcation of 5-hmC by
β-glucosyltransferase is independent of
DNA sequence or structural context2.

/

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)
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),e^
(''e^,''e^
Ceki[XhW_d:D7)%,«^

Figure 2: Real-time PCR analysis of the enrichment of
the 5-hmC DNA from mouse brain.
Mouse brain DNA was assayed at various quantities inthe Hydroxymethyl Collector Kit. Enriched DNA was
analyzed by real time PCR across multiple loci. The
enriched DNA was quantified and plotted as a percentage of the starting material. Kcnab1, Slc22a4 and Faim2
are methylated targets and Control 2 is unmethylated.

What are the advantages?
• Covalent labeling – ensuresaccurate capture of DNA fragments
containing hydroxymethylcytosine• Highly sensitive – technique issensitive enough to enrich for DNA
fragments containing as few as
two 5-hmC residues per strand• Simple procedure – can becompleted in less than 4 hours• Positive control – 5-hydroxymethylated DNA and real time PCRprimers are included to conﬁrm thesuccess of the enrichment reactions

To learn more about all of Active Motif’s 5-Hydroxymethylcytosine products,
please call or visit us at www.activemotif.com/hmc.
Product

FormatCatalog No.

Hydroxymethyl Collector™

25 rxns

55013

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March 2012 • volume 13 • number 1

MeDIP-Seq Services

NEW: MeDIP-Seq Now Offered by Active Motif Epigenetic Services
The reliable identiﬁcation of differential DNA methylation is important for researchers interested in biomarker
identiﬁcation, as well as for those trying to understand the basis of disease, drug mechanism of action orenvironmental inﬂuences on epigenetics. To help speed research in these areas, Active Motif now offers
MeDIP-Seq as an end-to-end, genome-wide epigenetic service to identify differentially methylated regions.
How does MeDIP-Seq work?
In MeDIP-Seq, a highly speciﬁc antibody
that recognizes 5-methylcytosine isused to immunoprecipitate sonicatedgenomic DNA, resulting in the enrichment of genomic regions that are
methylated. Because 5-methylcytosine
antibody binds only to methylated
cytosines in the context of single-stranded DNA, the DNA must be denatured
prior to immunoprecipitation. As a resultof denaturation the enriched DNA cannot be processed for Next-Gen sequencing using the typical sequencing librarygeneration protocols, as these require
adaptor ligation to double-stranded
DNA. This problem is circumvented
by ligating the Next-Gen sequencing
adaptors to genomic DNA prior to theimmunoprecipitation. Following MeDIP,
the enriched regions can be directly
ampliﬁed with Next-Gen sequencing
compatible primers. Unique alignmentof the sequence tags across the genome
reveals the regions of DNA methylation.
MeDIP-Seq advantages
Several methodologies exist that can
detect DNA methylation on a genomewide scale. Whole genome bisulﬁtesequencing is the most comprehensive,providing single base resolution acrossthe entire genome. However, it requires
sequencing of the entire genome, whichis extremely expensive. Reduced Representation Bisulﬁte Sequencing (RRBS)
also yields single base resolution, but it
only interrogates 10% of all CpGs andit is more heavily biased toward CpG-

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9f=?ibWdZ

ADCY1
p12.3

45,600,000

45,650,000

45,700,000

45,750,000

Figure 1: MeDIP-Seq data compared to CpG density and RRBS.
MeDIP-Seq was performed using 2 µg of DNA from human PBMCs. Methylation peaks were mapped across the genome andthe image is zoomed in to look at the ADCY1 gene. The middle panel shows good correlation of MeDIP signal (purple bars)
with CpG density (overlaid gray peaks) indicating that MeDIP covers most CpG sites across the ADCY1 gene. RRBS data isshown at the top as blue bars. and coverage is limited to only a few regions.

c13orf35
q34

113,290,000

113,295,000

113,300,000

113,305,000

113,310,000

113,315,000

Figure 2: Results from Active Motif’s MeDIP-Seq Service correlate well with published MeDIP results.
This image shows a 30 Kb region on chromosome 13. The top panel is data from a published, publicly available data set while
the bottom was produced by Active Motif’s MeDIP-Seq Service. Overlaid gray peaks show CpG density across the region.

rich regions than any other technique.
This is not necessarily an advantage as
most CpG islands are unmethylated,and because there is growing interestand focus on regions with lower CpG
density. MeDIP-Seq coverage is not as
biased toward high CpG density and intheory can interrogate all regions acrossthe genome. The expanded genomic
coverage of MeDIP-Seq compared to
RRBS increases the likelihood of identifying differentially methylated regions in
multi-sample studies (Figure 1).

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The MeDIP-Seq Service includes
The customer submits puriﬁed DNA,frozen tissues or cell pellets, then we:
1) Prepare the sample
2) Perform MeDIP with 5-mC mAb
3) Amplify the enriched DNA sample
4) Perform Next-Gen sequencing
5) Analyze the data
Other Services offered include• hMeDIP-Seq & ChIP-Seq• Bisulﬁte Sequencing• ChIP Antibody validation, & more

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5

Polycomb Proteins & Stem Cells

Polycomb Proteins Help Determine Embryonic Stem Cell Identity
Embryonic stem cells (ESCs) are cells derived from the inner cell mass of the developing pre-implantation embryothat gives rise to endoderm, ectoderm and mesoderm. ESCs are unique from all other cells types in that they candifferentiate into essentially all cell types in the developing and adult organism, a phenomenon termed pluripotency.
They also have the ability to divide into cells that have the same developmental state as the parent (self-renewal).
The ability of a cell to remain pluripotent and to self-renew requires the establishment of very complex gene regulation programs. Embryonic stem cells must facilitate the expression of genes required for self-renewal and pluripotency, but at the same time maintain all genes involved in lineage commitment and differentiation in a repressed state.
Unlimited developmental potential
Unlike the cells that are derived fromthem, embryonic stem cells by their very
nature have unlimited developmental
potential. Differentiated cells however,like a neuron or muscle cell, have lostthe ability (without intervention by the
researcher) to be anything other thanwhat they have become. Because ESCs
are genetically identical to all of thecells that are derived from them, theprimary difference between them is
not explained by genetics but rather by
epigenetics.
Stem cell identity
The Polycomb proteins are important
epigenetic determinants of stem cell
identity. They play an important role,
serving to help determine whether
stem cells remain pluripotent or commit
to a more speciﬁc cell fate. Originallyidentiﬁed in Drosophila as repressors ofgenes involved in body plan, Polycomb
proteins are segregated into one of two
protein complexes, Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2;
see Table 1). As some Polycomb complex subunits have multiple potential
component proteins (e.g. there exist
four isoforms of EED), various versionsof PRC1 and PRC2 complexes have been
identiﬁed.

EPIGENETICS NEWS6

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PRC1

PRC2

Protein

FunctionBMI-1, MEL18, MBLR, NSPC1

Unknown, required for self-renewal

CBX2/PC1, CBX4/PC2, CBX6,
CBX7 & CBX8/PC3

Chromodomain proteins, bind
H3K27me3

PHC1, PHC2, PHC3

Unknown

RING1, RING1b/RNF2

Monoubiquitylation of H2A K119

Protein

Function

EED (four isoforms)

Stimulation of Lys27 methylation

EZH2, EZH1

H3K27 HMTase

SUZ12

Unknown, required for ESC
differentiation

RbAp48

Histone chaperone

Table 1: Polycomb complexes and subunits.

Polycomb response elements
In ﬂies, Polycomb complexes are recruited to speciﬁc DNA sequences (termed
Polycomb Response Elements or PREs)
through the DNA binding protein Polyhomeotic (Ph). Although no mammalian
PRE sequences have been identiﬁed,
there are several potential DNA-binding
recruitment proteins, including OCT4,
YY1, JARID2, PCL2/MTF2 and AEBP2.
Non-coding RNAs, such as HOTAIR, havealso been implicated in the recruitmentof PRC2.

Once associated with chromatin, thePRC2 subunit EZH2 catalyzes trimethylation of histone H3 at Lys27 (H3K27me3;
see Figure 1). H3K27me3 has long been
known as a hallmark of regions ofrepressed chromatin. Trimethylation of
Lys27 leads to the recruitment of PRC1
through the binding of H3K27me3 bychromodomain-containing proteins in
PRC1. Interaction of PRC1 with nonadjacent regions of chromatin marked by
H3 Lys27 trimethylation may contributeto silencing through the establishment

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and keep up with the latest ﬁndings in the ﬁeld.

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March 2012 • volume 13 • number 1of a domain of specialized chromatin
structure that represses gene expression.
PRC1 also includes a histone modifying
enzyme, Ring1b, which catalyzes themonoubiquitylation of histone H2A at
Lys119 (H2AK119ub1). H2AK119ub1 has beenfound to repress transcript elongation by
RNA polymerase II.
The above recruitment and repression
model is more complex than described,
as many key regulatory genes in stem
cells are found in “bivalent” chromatin domains. These bivalent domains,which exhibit hallmarks of both active(H3K4me3) and repressed (H3K27me3)
chromatin, are thought to represent agene poised for either expression or
repression. Upon differentiation, bivalentdomains are resolved, depending upon
the resulting expression state. Genes
that are expressed in the target cell typemaintain histone H3 Lys4 methylation.
Markers of other cell types lose Lys4
methylation but retain the Lys27 methylation and thus go from poised to silent.
In addition to cell type-speciﬁc genes
and those involved in lineage commitment and differentiation, targets of Polycomb repression in stem cells include
tumor suppressors, to prevent cell senescence and potentially promote proliferation. Genes involved in apoptosis arealso repressed in a Polycomb-dependantfashion. In terminally differentiatedcells, Polycomb proteins participate
in the repression of pluripotency andstem cell-speciﬁc genes, as well as genes
expressed in other cell types.
Summary
It is essential to regulate stem cell
self-renewal and orchestrate the differentiation process, and it is clear that
Polycomb proteins are a crucial part ofwww.activemotif.com

Polycomb Proteins & Stem Cells

this regulation. Cells lacking EED or SUZ12
show aberrant expression of lineagespeciﬁc genes which interfere with
their ability to differentiate properly.
Differentiation is also hampered by theinability to repress pluripotency genes.

Mutations in Polycomb proteins are also
linked to the establishment of cancer,
as aberrant Polycomb function can
produce cancer stem cells that exhibit
inappropriate differentiation andperpetual self-renewal.

Figure 1: Simpliﬁed model of Polycomb Group (PcG)-mediated repression.
PRC2 is recruited to chromatin which allows trimethylation of histone H3 Lys27 by EZH2. PRC1 is then recruited to chromatinthrough the recognition of H3K27me3 by chromobox (CBX) proteins via the chromodomain. RNF2/RING1 homologs are
E3 ubiquitin ligases for H2A, which is monoubiquitylated at Lys119. The RYBP repressor protein recognizes H2A monoubiquitylation, contributing to transcriptional repression. Combined, these activities induce and maintain transcriptional
repression. Gray circular outlines depict other PRC subunits and associated proteins.

STEM CELL ANTIBODIES
Active Motif has a large and expanding list of antibodies to proteins important instem cell biology. Visit www.activemotif.com/stemcellabs for the complete list.
Product

FormatCatalog No.

BMI-1 antibody (mAb)

100 µg39993

CBX8 antibody (pAb)

100 µl61237

EED antibody (mAb)

100 µg61203

EZH2 antibody (mAb)

100 µg

39875

EZH2 phospho Thr345 antibody (pAb)

100 µl

61241

Histone H3 trimethyl Lys27 antibody (mAb)

100 µg

61017

PCL2 antibody (mAb)

100 µg61153

Phc1 antibody (mAb)

100 µg39723

Phc2 antibody (mAb)

100 µg39661

Ring1B antibody (mAb)

100 µg39663

Suz12 antibody (mAb)

100 µg39877

YY1 antibody (pAb)

100 µl39071

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7

Histone Acetylation & Deacetylation

Better Solutions for the Analysis of Histone Acetylation & Deacetylation
Histone acetylation and its antagonistic counterpart, histone deacetylation, play a major role in chromatin structureand gene expression. Acetylation of lysine residues on histone proteins leads to a less compact chromatin conﬁguration that is associated with gene expression. Acetylation of histones H3K9, H3K18, H4K12 and H4K16 are associatedwith diseased states and could be potential targets in therapeutic studies. To better understand the role of histone
acetylation and deacetylation on chromatin structure and gene expression, Active Motif offers assays, antibodies andenzymes for the analysis of these modiﬁcations.

HAT & HDAC Assays to Easily Screen for Changes in Histone Acetylation
Histone Deacetylase (HDAC) Assay Kit
For researchers interested in studying
histone deacetylation, Active Motif alsooffers both a colorimetric and ﬂuorescent HDAC Assay Kit. The kit contains anacetylated peptide substrate that can bedeacetylated by Class I, IIB and IV HDAC
enzymes. A deacetylated assay standard
is provided to enable calculation of
HDAC activity in pmol/min/mg.
To download a copy of our Histone
Analysis Products brochure, go towww.activemotif.com/info.

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How does the HAT Assay work?
The HAT Assay Kit is a quick and sensitive method to determine the activityof your own source of puriﬁed histone
acetyltransferase (HAT), or to screen forpotential inhibitors of HAT activity. The
ﬂuorescent assay includes both histone
H3 and H4 N-terminal histone tail substrates. HATs will catalyze the transfer ofacetyl groups from the provided acetylCoA to generate an acetylated peptide
and CoA-SH. After stopping the reaction
with stop solution, a developer is addedthat reacts with the free sulfhydryl
groups on CoA-SH to give a ﬂuorescentsignal (Figure 1). A standard curve can begenerated in order to relate the ﬂuorescence of your HAT to pmol/min/µg
speciﬁc activity.

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Figure 1: HAT inhibitor effects on p300 activity.
Fifty nanograms of the acetyltransferase p300 wereassayed per well with 50 µM acetyl-CoA and 50 µM
histone H3 or H4 peptide substrates in the absence orpresence of 15 µM anacardic acid, a known HAT inhibitor.
The background signal indicates the level of autoacetylation present from the p300 acetyltransferase.

Product

FormatCatalog No.

HAT Assay Kit (Fluorescent)1 x 96 rxns

56100

HDAC Assay Kit (Fluorescent)1 x 96 rxns

56200

HDAC Assay Kit (Colorimetric)

1 x 96 rxns

56210

Antibodies & Enzymes for the Study of Histone Acetylation
Need an active HAT or HDAC?
Whether you are looking for a control
enzyme to compare alongside your own
source of HAT or HDAC, or if you want
an enzyme for inhibitor screening, Active
Motif offers active recombinant proteinsfor the study of histone acetylation andhistone deacetylation.
• p300• GCN5• HDAC 1-9• SIRT1 & SIRT68North America 877 222 9543

Histone acetyl antibodies
Visit our website to see the complete
list of available histone antibodies.
• Large portfolio includes more than
40 antibodies speciﬁc to histone
acetylation modiﬁcations• Versatile target selection contains
antibodies for H2A, H2B, H3 & H4
acetylation targets• Validated applications include ChIP,
ChIP-seq, ChIP-chip, IF and WB• Sample size antibodies available

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Try our search ﬁlters to streamline yoursearch results
To browse the complete list of available
antibodies and enzymes, please visit
our website at www.activemotif.com.
Use our search ﬁlters on the left-handside of our web page to narrow yourresults based on, for example, antibody“Modiﬁcation” or recombinant protein“Protein Category”. (See page 9 for more
information on using the search ﬁlterson our website).

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March 2012 • volume 13 • number 1

Website Search & Filters

Navigate the Active Motif Website with the Greatest of Ease
Active Motif has recently added “Faceted Search” to its website. This enables you to perform very speciﬁc searches
using the Filters located in the left-hand column of every page. Multiple Filters can be applied at the same time andthey can also be combined with Search Terms, so you can ﬁnd the products you want faster. See below for examples.

Want to generate a list of antibodies
for a speciﬁc type of modiﬁcation?
To display a list of high-quality antibodies speciﬁc for acetylated proteins, theﬁrst step is to click on “Antibodies”
under Filters in the left column. This
expands “Antibodies” so that all subcategories that can be applied as Filters
are displayed. (The numbers shown inparentheses tell you how many products
are in each category; these will update
as you apply Filters.) One of the Filters is“Modiﬁcation”. Clicking on this expands“Modiﬁcation” so that it displays all
types of modiﬁcation-speciﬁc antibodies
available. Selecting “Acetylated” will takeyou to a Search Results page in which
the Antibodies tab shows all of ourantibodies against acetylated targets.
(As we also offer Kits and RecombinantProteins that meet the search criteria
for “Acetylated”, tabs for these are also
displayed above the results chart.)

www.activemotif.com

Search Results can be further narrowed
by applying additional Filters. For example, under “Application” you could clickon “Chromatin Immunoprecipitation”,
which would then update the results sothey display ChIP-validated antibodies
against acetylated proteins. (Note thatthe tabs for Kits and RecombinantProteins disappear, as no products of
those types meet both search criteria.)
Combine Search Terms with Filters
You can also apply Keyword searches incombination with one or more Filters.
In our current example, if you type “H3”in the Search Terms box then click “Go”,
the Search Results will become evenmore speciﬁc. Now, only ChIP-validated
Histone H3 antibodies that are against
acetylated proteins are found. Filters cancontinue to be applied until the foundresults cannot be narrowed any further.

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Filters will disappear if they will notchange the Results. For example, if you
have generated a Search Result in which
all antibodies are Polyclonal, “Antibody
Type” will longer be displayed as a Filter
as applying it won’t change the results.
Removing Filters is also useful
Finally, Applied Filters can be removed inany order by clicking the “x” next to its
name in the gray bar above the results.
To demonstrate, click on the “x” next to“Chromatin Immunoprecipitation”. The
Search Results now display products
under the Antibodies, Kits and Recombinant Proteins tabs that meet the “H3”and “Acetylated” search criteria.
Find what you’re looking for faster
Faceted Search enables you to quickly
ﬁnd the speciﬁc products you’re looking
for. Try it today at www.activemotif.com.

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9

Super Resolution Microscopy

NEW: Antibodies and Reagents for Super-resolution GSDIM Microscopy
Until recently, ﬂuorescence microscopy has been limited by the level of resolution that can be achieved in imaging
experiments. The current diffraction limit is approximately 200 nm and does not allow the evaluation of the nanoarchitecture of the cell or the detection of cellular events in molecular detail. Ground State Depletion with Individual
Molecule return (GSDIM) is one of the super-resolution techniques that has been developed to overcome theselimitations. GSDIM provides the ability to image structures as small as 20 nm. These improvements in microscopy
enable scientists to decipher the nanostructure of the cell in details that previously could not be resolved (Figure 1).
In regular ﬂuorescence microscopy,
diffraction limits image resolution toroughly half the wavelength of theemitted light. Individual ﬂuorescent dye
molecules often cannot be opticallyseparated as they are too close. In Figure
2A, the dyes in the excitation spot will
overlap and appear to be one signal.
Super-resolution by localization
In GSDIM, the majority of dye molecules
within the excitation spot are transformed into a dark state; only some
single dye-molecules, which are well
separated from one another, are able toemit ﬂuorescence (Figure 2B). After thesemolecules are imaged with a camera, amathematical algorithm determines thelocations of the next set of individual
dye molecules that will be allowed to
ﬂuoresce, and another image is created(Figure 2C). This is repeated thousandsof times. By overlaying all of the images,
a high-resolution image of the sample isconstructed. With GSDIM, a resolutionof 20 nm is possible.

Figure 1: Active Motif’s ﬂuorescent secondary
antibodies in GSDIM microscopy.
HeLa cells were stained with alpha Tubulin mouse mAb(Clone 5-B-1-2) and Chromeo™ 505 Goat anti-mouse
IgG (Catalog No. 15030). The widefield image (left) and
the GSDIM image (right) are provided courtesy of Leica
Microsystems, Germany.

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North America 877 222 9543

7$

8$

9$

Figure 2: The principle of GSDIM microscopy.
Illustration of the exposed area of a sample in classical widefield microscopy (A) or in GSDIM microscopy (B + C) in which
only a small number of spatially separated, single dye molecules are allowed to emit fluorescence at the same time. The
spheres represent individual dye molecules in fluorescent (green) or “off” mode (black).

In contrast to other stochastic methods,
GSDIM has the advantage that regular,
high-quality ﬂuorescent dyes can beused, which allows researchers to workwith their regular staining protocols.
Accordingly, Active Motif’s IF-validated
primary antibodies, high-quality
Chromeo™-, ATTO- and Rhodamine
6G-conjugated secondary antibodies
and ﬂuorescent Chromeo Dyes will help
researchers take advantage of the new,powerful GSDIM technology, ensuring
the highest quality images possible.

Advantages of Active Motif’s dyes andsecondary antibodies for use insuper-resolution microscopy• Brilliant• Stable• Low background• Optimized conjugation protocols• Recommended by Leica for use
with its SR GSD microscope
For more information on GSDIM and our
ﬂuorescent dyes and secondaries, please
visit us at www.activemotif.com/gsdim

Product

FormatCatalog No.

Chromeo™ 488 Goat anti-Mouse IgG

1 mg

15031

Chromeo™ 488 Goat anti-Rabbit IgG

1 mg

15041

Chromeo™ 505 Goat anti-Mouse IgG

1 mg

15030

Chromeo™ 505 Goat anti-Rabbit IgG

1 mg

15040

Chromeo™ 546 Goat anti-Mouse IgG

1 mg

15033

Chromeo™ 546 Goat anti-Rabbit IgG

1 mg

15043

ATTO 532 (GSD) Goat anti-Mouse IgG

250 µl

15070

ATTO 532 (GSD) Goat anti-Rabbit IgG

250 µl

15072

Rhodamine 6G (GSD) Goat anti-Mouse IgG

250 µl

15074

Rhodamine 6G (GSD) Goat anti-Rabbit IgG

250 µl15076

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com

March 2012 • volume 13 • number 1

Chromatin IP Controls & Primers

NEW: ChIP-IT® Control Kits for Real Time PCR Analysis
Optimizing chromatin immunoprecipitation (ChIP) experiments to conﬁrm the quality of your chromatin preparation
or validate your antibody for use in ChIP can be difﬁcult without the right controls. To make it easier to determine if
your ChIP reactions are working, Active Motif offers a variety of ChIP kits and accessories including control chromatin, ChIP-validated antibodies, control qPCR primer sets and our new species-speciﬁc control kits for use in real time
PCR analysis. To see a full listing of available ChIP kits and accessories, please visit www.activemotif.com/chip.
ChIP-IT® Control qPCR Kits
In an effort to provide more complete
chromatin analysis, Active Motif islaunching our new ChIP-IT® Control
qPCR Kits for human, mouse and rat.
Control kits are ideal for ChIP antibody
validation when run in parallel with yourantibody of interest. The control kit will
conﬁrm the chromatin preparation and
IP procedure worked properly and allow
you to assess your test antibody.
One of the advantages of using the
ChIP-IT Control qPCR Kits is that each
kit contains both positive and negative
control PCR primer sets. This enables adirect comparison of gene targets forthe same antibody and eliminates thevariation that can be observed whentrying to analyze ChIP results against thenegative control IgG, as each antibody
has a different binding afﬁnity. All of ourcontrol qPCR primer sets have been rigorously tested by our Epigenetic Services
division and validated to work for you.

What is included in the Control Kits?
• RNA pol II antibody – positive
control antibody to verify the ChIP
reaction was successful• Bridging antibody – enhances thebinding afﬁnity of mouse monoclonal antibody to protein G beads
and is recommended for use withall mouse monoclonal antibodies,including the RNA pol II control• Negative control antibody – mouse
IgG antibody that can be used toevaluate non-speciﬁc binding• Positive control qPCR primers –species-speciﬁc control primer set
that ampliﬁes the RNA pol II ChIP
• Negative control qPCR primers –species-speciﬁc control primer set
not ampliﬁed in the RNA pol II ChIP

Save 30% on new Control Kits
To help you get your ChIP experiments started with suitable controls,
for a limited time Active Motif isoffering a 30% discount on all of
our new ChIP-IT Control qPCR Kits
for human, mouse and rat when you
purchase one with a ChIP-IT® Express
or ChIP-IT® Express Enzymatic Kit.
Use the control kits in combination
with either of our ChIP-IT Express
Kits, which provide a detailed
protocol and optimized reagents for
successful chromatin preparation andimmunoprecipitation. Simply cite thepromotion code “MV13-1” when you
order to receive your discount.

Product

FormatCatalog No.

ChIP-IT® Control qPCR Kit – Human

5 rxns53026

ChIP-IT® Control qPCR Kit – Mouse

5 rxns

53027

ChIP-IT® Control qPCR Kit – Rat

5 rxns53028

ChIP-IT® Express

25 rxns

53008

ChIP-IT® Express Enzymatic

25 rxns

53009

Expansive Offering of Species-validated Control qPCR Primer Sets for ChIP
Need ChIP controls for your species?
Active Motif offers human, mouse, rat,
Zebraﬁsh, Drosophila and yeast qPCR
primer sets for use as positive andnegative controls for many of the morecommon ChIP targets, including histone
modiﬁcations, transcription factors andmethylated DNA (Table 1). To see the fulllist of over 35 available primer sets andtheir validated targets, visit our website
at www.activemotif.com/chipprimers.

www.activemotif.com

Product

Use as a Control for:

Catalog No.

Human Positive Control Primer Set ACTB-2

Many activating histone marks

71005

Human Positive Control Primer Set MYT1

H3K27me3 & EZH2

71007

Human Positive Control Primer Set ZC3H13

5-mC (MeDIP)

71009

Human Negative Control Primer Set 1

Most TFs & Histone mods, Pol II & 5-mC

71001

Mouse Positive Control Primer Set Hoxc10

H3K27me3 & EZH2

71019

Mouse Negative Control Primer Set 2

Most TFs & Histone mods, Pol II & 5-mC

71012

Drosophila Positive Control Primer Set Act5C

RNA Pol II phospho Ser2

71030

Drosophila Negative Control Primer Set 1

Most TFs & Histone mods, & Pol II

71028

Table 1: A few examples of the many species-speciﬁc ChIP Control qPCR Primer Sets offered by Active Motif.

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