Confocal

STEDHigh Resolution STED Microscopy Products (page 11)
IN THIS ISSUE

DNA Methylation, Sample Preparation & Antibodies
2 5-Hydroxymethylcytosine: A New Analysis
in DNA Methylation

8 NEW: Dounce Homogenizer to Assist in
Cell and Chromatin Preparations

3 NEW: Biotin-based Enrichment of
5-Hydroxymethylcytosine

9 NEW: Protease Inhibitor Cocktail for
Better Sample Preparation

4 NEW: Enzymes for the Study of
5-Hydroxymethylcytosine

9 Nuclear Extraction Kit for Nuclear,
Cytoplasmic or Whole-cell Lysates

5 NEW: MeDIP Enrichment of 5-Methylcytosineor 5-Hydroxymethylcytosine

10 Antibodies to Stem Cell Regulators toFurther your Stem Cell Research

6 NEW: ChIP qPCR Primers and Controls
Ensure Your Experiments Work Every Time

10 Highly Validated Antibodies and ELISAs
for the Study of Nuclear Receptors

7 NEW: Multi-Sample Sonicator, Probe
Sonicator and Cooled Sonication Platform

11 Antibodies and Reagents forHigh Resolution Confocal
STED Microscopy

8 DNMT Assay to Analyze Changes in
CpG Methylation

5-Hydroxymethylcytosine DNA Methylation

5-Hydroxymethylcytosine: A New Analysis in DNA Methylation
In mammals and other vertebrates, DNA methylation usually occurs at the C5 position ofcytosines (5-mC), mostly within
CpG dinucleotides. Normally, CpG
islands, short CG-rich regions, areunmethylated; in cases where
methylation does occur, the associated gene is silenced. Aberrant
methylation of these CpG regions
is often associated with disease.
In 2009, Kriaucionis and Heintz as
well as Tahiliani et al. discoveredthat a novel form of DNA methylation, 5-hydroxymethylcytosine(5-hmC), exists in mammals, and iselevated in neurons and embryonicstem cells. 5-Hydroxymethylcytosine results from the enzymaticconversion of 5-methylcytosine
into 5-hydroxymethylcytosine by
the TET family of cytosine oxygenases (Figure 1). To date, little isunderstood about the functional
relevance of 5-hmC in the mammalian genome.
One of the difﬁculties in studying
5-hmC is the fact that many of the
traditional techniques employed

NH2

OH

NH2

N

N
TET1, TET2, TET3

N
H

O

O

N
H

5-methylcytosine

5-hydroxymethylcytosine

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

to study DNA methylation, suchas bisulﬁte conversion or certain
methylation-sensitive restriction enzymes, do not distinguishbetween 5-mC and 5-hmC residues. Methyl CpG binding protein
enrichment methods only serve
to selectively bind 5-mC DNA
methylation, but cannot be used
to enrich for 5-hmC. In an effort
to better analyze the location andfunction of 5-hydroxymethylcytosine, new tools and techniques willneed to be employed.
Active Motif is pleased to offer
antibodies, assays and enzymes
for the direct analysis of 5-hydroxymethylcytosine. We werethe ﬁrst company to provide a
5-hydroxymethylcytosine antibody
that could be used for the detection and immunoprecipitationof 5-hmC DNA. We have since
expanded our product offering
to include enzymes capable ofdistinguishing between 5-mC and5-hmC residues, and afﬁnity enrichment kits to selectively isolate
5-hydroxymethylcytosine DNA.
Additional product details areavailable within this newsletter. To
learn more about all of the 5-hmC
products available, please give us acall or visit us on our website atwww.activemotif.com/hmc.

Save 10% on reagents to study 5-Hydroxymethylcytosine
For a limited time, get 10% off select products for 5-hmC.
For complete details and a list of eligible products,
please visit www.activemotif.com/promo.

2

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October 2011 • volume 12 • number 2

5-Hydroxymethylcytosine Enrichment

NEW: Biotin-based Enrichment of 5-Hydroxymethylcytosine
To better interrogate the functional relevance of 5-hydroxymethylcytosine (5-hmC) DNA methylation, Active Motif
has developed an assay kit that enables detection and afﬁnity enrichment of DNA fragments containing the5-hydroxymethylcytosine residue. The Hydroxymethyl Collector™ kit utilizes a β-glucosyltransferase enzyme totransfer a modiﬁed glucose moiety to 5-hydroxymethylcytosine residues in double-stranded DNA. This modiﬁed
glucose is then used to chemically attach a biotin conjugate for capture and enrichment with magnetic streptavidin
beads. The enriched DNA can then be analyzed by PCR, microarray or sequencing to better understand the role of
5-hydroxymethylcytosine in mammalian genomes.
How does Hydroxymethyl Collector™
isolate 5-hmC DNA?
First, genomic DNA is fragmented toa size range of 100-500 base pairs. The
DNA is then incubated in the presenceof a β-glucosyltransferase enzyme anda modiﬁed UDP-Glucose donor. The
enzyme transfers the glucose to 5-hydroxymethylcytosine residues, creating
glycosyl-5-hydroxymethylcytosine.

Following puriﬁcation, with the included
puriﬁcation reagents, the DNA is readyto use in PCR, microarray or sequencing.
What’s in the box?
The Hydroxymethyl Collector Kit contains all the reagents needed to performthe glucosylation reaction, biotin conjugation, streptavidin capture, elution andpuriﬁcation of 5-hydroxymethylcytosine
DNA. A positive control hydroxymethylated DNA standard and PCR primers arealso included in the kit to conﬁrm thesuccess of the enrichment reactions.
ReferencesSong et al. (2010) Nature Biotechnology 29: 68-72.

www.activemotif.com

Figure 1: Human tiling array using DNA enriched with Hydroxymethyl Collector.
Human brain DNA that was enriched using the Hydroxymethyl Collector Kit was amplified by whole genome amplificationand hybridized to Affymetrix Human Tiling 2.0R Array A containing chromosomes 1 and 6. The image shows a 1.2 million
base pair view of chromosome 6 where there is a clear enrichment of 5-hmC across the entire length of the JARID2 gene.

What are the advantages?
• Covalent labeling of 5-hmC ensuresaccurate capture of DNA fragments
containing hydroxymethylcytosine• The biotin-streptavidin capturesystem enables the use of stringent
binding and wash conditions• The technique is sensitive enoughto enrich DNA fragments containing
as few as two 5-hmC residues• Simple procedure can be completed
in less than 4 hours• Enriched DNA is suitable for use inanalysis of individual loci by PCR, or
whole-genome analysis by sequencing or microarray (Figures 1 & 2)

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Click chemistry is used to introduce
a biotin conjugate onto the modiﬁed
glucose. Magnetic streptavidin beads
and the included bar magnet are used
to capture the biotinylated 5-hmC DNA
fragments. The elution buffer releases
the DNA fragments from the biotin
conjugation, leaving you with DNA thatis enriched for 5-hmC methylation.

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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. This graph shows theHydroxymethyl Collector Kit has a range of detection
from 5 ng to 2.5 µg. The Mouse Negative Control Primer
Set 2 (Cat. No. 71012) primer served as a negative control.

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.

Price ($US)

Hydroxymethyl Collector™

25 rxns

55013

375

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North America 877 222 9543

3

5-Hydroxymethylcytosine Enzymes

NEW: Enzymes for the Study of 5-Hydroxymethylcytosine
Active Motif offers enzymes to study not only the mechanism of the conversion of 5-methylcytosine (5-mC) into
5-hydroxymethylcytosine (5-hmC), but also enzymes that enable differentiation between these two forms of DNA
methylation. These enzymes help to overcome some of the technical limitations that previously existed in theanalysis of 5-hydroxymethylcytosine.
β-Glucosyltransferase enzyme for
5-hmC modification
For researchers looking to study overallchanges in 5-hydroxymethylcytosine,
Active Motif offers separately the same
β-Glucosyltransferase enzyme includedin the Hydroxymethyl Collector™ Kit (seepage 3). This enzyme can modify the
5-hmC residue with the addition of a
glucose moiety. Following glucosylation,
the 5-hmC residues will be protectedfrom glucosyl-sensitive restriction
enzymes, such as Msp1 or Gla1. Alternatively, if the β-Glucosyltransferase
enzyme is used with a radiolabeled UDPglucose donor, it enables direct labelingof hydroxymethylated residues.
The β-Glucosyltransferase enzyme isprovided with a 10X reaction buffer, a
UDP-Glucose (uridine diphosphoglucose)
donor and 1 M DTT. One unit of enzyme
is deﬁned as the amount of enzyme required to fully protect 0.5 µg of a 5-hmC
DNA standard in 1 hour at 37°C in a total

Figure 1: Protection of 5-hmC by glucosylation.
A 5-hydroxymethylated DNA Standard (0.5 µg) was
incubated in the absence or presence of 1 unit ofβ-Glucosyltransferase for 1 hour at 37°C. Following column purification, reactions were treated with 4 units of
glucosyl-sensitive restriction enzyme, Gla1.

4

North America 877 222 9543

reaction volume of 50 µl from degradation by 4 units of a glucosyl-sensitive
restriction enzyme, as shown in Figure 1.
The 5-hmC methylated DNA standard
tested is a 338 base pair oligonucleotide
containing 122 hydroxymethylcytosine
residues. For more details on Active
Motif’s Methylated DNA Standards(Catalog No. 55008), please visit our
website at www.activemotif.com/hmc.
Tet1 for 5-hmC conversion assays
Active Motif offers a recombinant Tet1
protein for use in 5-hmC conversion assays. Tet1 is a member of the TET familyof cytosine oxygenases that convert
5-methylcytosine into 5-hydroxymethylcytosine. This enzyme can be used
to convert 5-mC containing DNA into
5-hmC containing DNA (Figure 2). To
learn more, please visit our website.

Figure 2: Recombinant Tet1 activity assay.
Double-stranded DNA containing 5-methylcytosine
was incubated with 5 µg of recombinant Tet1 enzyme(+Tet1) or without Tet1 enzyme (-Tet1). These samples
and an unmethylated DNA control (DNA) were thenspotted onto a nylon membrane and incubated with
5-Hydroxymethylcytosine antibody (Catalog No. 39769)
to detect the conversion of 5-methylcytosine into
5-hydroxymethylcytosine.

PvuRts1I enzyme for direct digestionof hydroxymethylated DNA
While glucosyl-sensitive restriction enzymes offer a method to enzymaticallydifferentiate between 5-mC and 5-hmC,
they require the prior addition of a glucose moiety to the 5-hydroxymethylcytosine residue. Active Motif’s novel new
PvuRts1I restriction enzyme, however,is able to bypass this intermediary stepand directly differentiate between 5-mC
and 5-hmC residues. PvuRts1I directly
cleaves hydroxymethylated DNA in its
non-glucosylated form, but will not digest 5-methylcytosine or unmethylated
cytosine residues (Figure 3). The enzyme
also cleaves glucosylated-5-hmC DNA,but at a lower efﬁciency. To learn more
about this unique enzyme’s digestion
properties, please visit our website anddownload the data sheet.

Figure 3: PvuRts1I enzyme digests 5-hmC DNA.
One µg of unmethylated (Un), 5-methylcytosine (mC)or 5-hydroxymethylcytosine (hmC) Methylated DNA
Standard (Catalog No. 55008) was incubated in theabsence or presence of 1 unit PvuRts1I restriction
enzyme for 30 minutes at 22°C. Each reaction was run
on a 2.5% agarose gel alongside a 1 kb DNA ladder. Onlyhydroxymethylated DNA was digested in the presenceof the PvuRts1I restriction enzyme.

Product

FormatCatalog No.

β-Glucosyltransferase enzyme

500 Units

55012

275

PvuRts1I restriction enzyme

50 Units

55011

225

Recombinant Tet1 protein, active

25 µg

31363

320

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

Price ($US)

www.activemotif.com

October 2011 • volume 12 • number 2

MeDIP & hMeDIP Assays

NEW: MeDIP Enrichment of 5-Methylcytosine or 5-Hydroxymethylcytosine
Active Motif now has two kits available for the enrichment of methylated DNA via antibody immunoprecipitation.
Methylated DNA immunoprecipitation (MeDIP) is an immunocapture technique in which an antibody speciﬁc formethylated cytosines is used to immunoprecipitate methylated genomic DNA fragments. The hMeDIP Assay uses
a highly speciﬁc, puriﬁed 5-hydroxymethylcytidine antibody to selectively enrich for DNA fragments containing
5-hydroxymethylcytosine methylation from the rest of the genomic DNA population, while the MeDIP Assay uses a
monoclonal 5-methylcytidine antibody to selectively enrich for traditional 5-methycytosine DNA methylation.

For added convenience, the kit also
includes a negative control mouse IgG
antibody, positive control MseI digested
human genomic DNA and real time PCRprimers that can be used to verify the
efﬁciency of the MeDIP enrichment.
How does the hMeDIP Assay differ?
One of the ways the hMeDIP Assaydiffers from the MeDIP Assay isthat it does not require the use of
single-stranded DNA fragments. The
5-hydroxymethylcytidine antibody usedin the hMeDIP kit is capable of recognizing both double-stranded and singlestranded 5-hmC methylated fragments.
This is advantageous because the eluted
DNA will be double-stranded, which can
bypass problems associated with linker
bias when preparing enriched DNA for
downstream Next-Gen sequencing.
Another difference is the hMeDIP Kit
uses a polyclonal antibody, therefore,

www.activemotif.com+'*,,$d\k_pcZpk`[`e\d8Y*'f]@eglk

How does the MeDIP Assay work?
Active Motif’s new MeDIP Assay utilizes
a highly speciﬁc monoclonal antibody
that recognizes 5-methylcytosine
residues on single-stranded DNA to
immunoprecipitate and enrich formethylated DNA fragments. The
5-methylcytidine antibody is added in
the presence of a bridging antibody andprotein G magnetic beads. Following
an overnight incubation, the included
magnet is used to collect the beads forwashing and elution.

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JXdgc\(JXdgc\)N`k_Yi`[^`e^Xek`Yf[pJXdgc\(JXdgc\)N`k_flkYi`[^`e^Xek`Yf[pFigure 1: Real time PCR results using the MeDIP Assay.
Mse I digested human genomic DNA (500 ng) was processed in duplicate using the MeDIP Assay. Reactions
contained either the 5-methylcytidine monoclonal
antibody or the negative control mouse IgG in the presence or absence of bridging antibody. Eluted DNA was
purified and tested in real time PCR with the included
ZC3H13 PCR primer mix. The methylated ZC3H13 locus
is specifically enriched in the IP reactions using the
5-methylcytodine antibody and bridging antibody, while
reactions that lacked bridging antibody, or contained
the negative control mouse IgG showed no enrichment.

it does not require the assistance of abridging antibody for complete captureby the protein G magnetic beads. Finally,
the hMeDIP kit includes three methylated DNA standards (unmethylated,
5-methylcytosine and 5-hydroxymethylcytosine) for use as spike controls in the
IP reaction to determine efﬁciency.

Antibodies for methylation analysis
For researches interested in detectionof methylated DNA instead of enrichment by MeDIP and hMeDIP Assay Kits,
Active Motif also offers the antibodies
separately. For 5-hydroxymethylcytosine
detection, researchers have the optionof the puriﬁed polyclonal antibody thatis used in the hMeDIP kit, the unpuriﬁed
polyclonal whole rabbit serum or our
exclusive monoclonal antibody, Clone
59.1. For 5-methylcytosine detection,
both the monoclonal 5-methylcytidine
and Bridging antibodies that are includedin the MeDIP Kit are available separately.
5-Hydroxymethylcytosine Services
In addition to our reagents and assays,
Active Motif also offers Epigenetic
Services for the analysis of 5-hydroxymethylcytosine. Our service offerings
include hMeDIP-seq and hMeDIP-chipfor genome-wide analysis of hydroxymethylation. This enables you to takeadvantage of Active Motif’s expertise
without having to be an expert yourself.
Visit www.activemotif.com/services tolearn about our Epigenetic Services.

Product

FormatCatalog No.

MeDIP

10 rxns

55009

350

hMeDIP

10 rxns

55010

350

5-Hydroxymethylcytidine antibody mAb (Clone 59.1)

100 µg39999

330

5-Hydroxymethylcytidine antibody pAb

100 µl39769

330

5-Hydroxymethylcytidine antibody pAb (IgG)

100 µg39791

330

5-Methylcytidine antibody mAb (Clone 33D3)

50 µg

39649

330

Bridging Antibody for Mouse IgG

500 µg

53017

75

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Europe +32 (0)2 653 0001

North America 877 222 9543

Price ($US)

5

Chromatin IP Accessories

NEW: ChIP qPCR Primers and Controls Ensure Your Experiments Work Every TimeThe number of variables in ChIP experiments makes them extremely complex, from ﬁxation to cell type tosonication to antibody incubation, enrichment and analysis. To make your ChIP experiments more successful,
we have developed a line of accessory kits and reagents designed to make it easier for you to troubleshootyour ChIP experiments and to validate your results.

Control qPCR Primer advantages• Available for many popular
ChIP targets• Both positive and negative controls
offered for a variety of species• Tested on multiple cell lines• Supplied in solution and ready to use

6

North America 877 222 9543),

8_dZ_d];l[dji:[Yj[Yj[Z%'&&&9[bbiChIP Control qPCR Primer Sets
The proper analysis tools are crucial forcarrying out successful chromatin IP. The
correct positive and negative control
PCR primers are essential to determine
the success or failure of your experiment. Active Motif offers a large varietyof species-speciﬁc qPCR primer sets foruse as positive and negative controls formany of the more common ChIP targets,
including histone modiﬁcations, transcription factors and methylated DNA.
Use of our human, mouse, rat, Zebraﬁsh,
Drosophila and yeast primer sets willsave you the time and effort required
to design, synthesize and test your own
species- and gene-speciﬁc control primers. Our qPCR primer sets are used regularly by our Epigenetics Services divisionand so have been rigorously tested andvalidated to work for you. When usedin combination with our ChIP-validated
antibodies and Ready-to-ChIP Chromatin, you have all the tools you need forsuccessful chromatin IP. To see the manydifferent primer sets available, go towww.activemotif.com/chipprimers.

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Figure 1: ChIP of RNA pol II CTD phospho Ser5 mAb.
ChIP was performed using 10 µg of chromatin fromhuman LP1 cells and 10 µg RNA pol II CTD phospho Ser5
antibody (Catalog No. 61085). ChIP DNA was used in
qPCR with Active Motif’s human control qPCR primer
sets, as indicated. Data are presented as Binding Events
Detected per 1000 Cells.

ChIP-IT™ Control Kits
Chromatin immunoprecipitation is a
DNA enrichment technique, not puriﬁcation, so ChIPs are unavoidably contaminated with non-speciﬁc chromatin. This
often leads to false PCR products thatcan make data difﬁcult to interpret andcomplicate the validation of antibodies
and primer sets. To solve these problems, we offer species-speciﬁc ChIP-IT™
Control Kits that provide positive andnegative control antibodies, a positive
control PCR primer set and a PCR buffer/
DNA loading dye that makes your PCR
reactions gel-ready.

Ready-to-ChIP Chromatin
For your convenience, Active Motif offers its Ready-to-ChIP chromatin from anumber of ENCODE cell lines that havebeen optimally sheared by sonicationand validated in ChIP for inclusion as
a positive control or test sample. As aresult, you can more easily validate yourown antibodies and primer sets. The
chromatin can be used with all of the
ChIP-IT Express Kits and controls, so you
can be certain that the only variable invalidating a new antibody for ChIP is theantibody itself.
ChIP-IT Express Shearing Kits
Designed to work speciﬁcally with ourChIP-IT Express Kits, the sonication andenzymatic shearing kits give you thesame shearing components as thosefound in ChIP-IT Express Kits, but ingreater quantities to allow you to optimize your shearing conditions before
proceeding with ChIP experiments.
For more information about ouraccessories and industry-leading ChIP
kits, visit www.activemotif.com/chip.

Figure 2: Genomic Localization of Human Positive Control Primer Set GAPDH-2.
Image representing the relative location of the Human Positive Control Primer Set GAPDH-2 primer set amplicon within
the genome, as generated by the UCSC Genome Browser. The top image represents the location on chromosome 12, thebottom image represents the amplicon relative to the GAPDH gene.

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October 2011 • volume 12 • number 2

Sonication Equipment

NEW: Multi-Sample Sonicator, Probe Sonicator and Cooled Sonication Platform
Sonication is frequently used to prepare sheared chromatin for ChIP, as well as for standard cell disruption, DNA/RNA
shearing and other homogenization applications. Active Motif is pleased to introduce its EpiShear™ line of sonication
products, which will save you time and effort while ensuring that you obtain more reproducible results.
Programmable generators – why notchoose complete control?
Both the EpiShear™ Multi-Sample
Sonicator and the EpiShear™ Probe
Sonicator are controlled by microprocessor units that offer both programmableand manual operation. Each has a keypadand digital display that make it easy toprogram the amplitude and duration of
On and Off pulse cycles. Pulse intensity
to be set from 20-100%, enabling you tochoose exact parameters for your celltype. The digital display shows the total
elapsed time, and provides real-timeenergy monitoring of both wattage andjoules. These features make it easier toobtain more reproducible results when
you perform subsequent sonications.

Figure 1: The EpiShear Multi-Sample Sonicator / Chiller.
The EpiShear Multi-Sample Sonicator / Chiller includes a powerful 750-watt generator with a keypad and digital display thatmake it easy to program and monitor sonication. The unit’s cup horn sonicator is housed in a compact sound enclosure to
reduce sonication noise. The cup horn can process up to 8 samples simultaneously, which rotate continuously to ensure all
samples are processed equally. It is mounted in an acrylic bath that is filled with water. The bath and enclosure are plumbedwith quick-connect hoses that attach to a thermoelectric chiller, which keeps the samples at 2-4°C during sonication.

Process up to 8 samples at once
The Multi-Sample Sonicator is a high-intensity cup horn sonicator. Its 750 wattsof power enable continuous operation
to shear even the most problematic
samples, like T cells. The unit can process
up to eight 1.5 ml vial samples at once,and comes with a sound enclosure and athermoelectric chiller (Figure 1). For more
complete information, please visit us atwww.activemotif.com/cuphorn.
Sonicate large or small samples
While the EpiShear™ Probe Sonicator is
a compact, economical unit (Figure 2), it
is still a fully programmable unit with all
the features that will help ensure yourreproducible results. It is supplied witha 1/8" microtip probe that enables you
to shear one 500 µl to 15 ml sample at atime. Other size probes are available that
expand its range from 200 µl to 50 ml.
For more complete information, please
visit www.activemotif.com/probe.

www.activemotif.com

Figure 2: The EpiShear Probe Sonicator and the EpiShear Cooled Sonication Platform.
The EpiShear Probe Sonicator is a compact 120-watt unit that can process small or large samples. The EpiShear Cooled
Sonication Platform, sold separately, precisely positions the probe every sonication, greatly enhancing sample-to-sample
reproducibility. The tube coolers, available in 3 sizes, eliminate the need to rest the sample in an ice bath between pulses.

Make any probe sonicator easier to use
The EpiShear Cooled Sonication Platform,
affectionately known as “Big Jack”,
greatly increases reproducibility byenabling you to precisely position thedepth of the probe in your sample every

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time. It can be used in a sound enclosure
or on a base. Coolers that ﬁt microfuge,
15 ml and 50 ml tubes keep your sample
cold during sonication, so you don’t have
shuttle it to and from an ice bucket. See
www.activemotif.com/platform.

North America 877 222 9543

7

CpG Methylation Analysis and Sample Preparation

DNMT Assay to Analyze Changes in CpG Methylation
DNA methylation usually occurs at the ﬁfth carbon of cytosine, within the context of a CpG dinucleotide. Modiﬁcations at these sequences by DNA methyltransferase enzymes (DNMTs) can have profound effects on transcription.
Active Motif’s DNMT Activity / Inhibition Assay is a fast, user-friendly assay to simplify the measurement of DNA
methyltransferase activity or the effects of inhibitor compounds without the need for radioisotopes.
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Non-radioactive DNMT Assay
The DNMT Activity / Inhibition Assay
provides all the reagents needed to
study DNA methyltransferase activity
from recombinant DNMT enzymes or
nuclear extract samples. The sensitive ELISA-based method is uniquein that it utilizes a methyl-CpG binding domain (MBD) protein to detect
methyltransferase activity, instead ofa radioactively labeled methyl-donor.
MBD proteins are capable of binding
methylated DNA with a high afﬁnity,
which increases the sensitivity of theassay and enables detection from as
little as 0.5 ng puriﬁed DNMT enzyme
or 0.5 µg nuclear extract. In addition toscreening for DNMT activity, the assay
can also be used to screen for changes in
CpG methylation following treatment by
DNA methylation inhibitors (Figure 1).

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C9<#-Y[bbi!%#/,^ekhFheYW_d[jh[Wjc[djFigure 1: DNMT inhibition of MCF-7 cells with procaine.
The DNMT Activity / Inhibition Assay was used toscreen for DNMT inhibition in MCF-7 cells that were
either untreated or treated with procaine for 96 hours.
Nuclear extracts were prepared using Active Motif’s
Nuclear Extract Kit (see page 9) and 5 µg of each condition was assayed with a 1.5 hour incubation time and a
3 minute developing time. The 5 mM procaine treatment
showed 43% inhibition of DNMT activity as compared tothe mock treated samples.

DNMT Activity Assay advantages• Non-radioactive – colorimetricassay is easily quantiﬁed on a
microplate reader at 450 nm• Sensitive – unique MBD protein
approach enhances the sensitivityof detection from either puriﬁedproteins or nuclear extracts• Fast – assay can be completed inless than 3 hours• Flexible – stripwell plate allows
screening of 1 to 96 samples• Versatile – works with DNMT1,
DNMT3a & DNMT3b proteins
To see all the available methylation kits,
visit www.activemotif.com/dnamt.

Product

FormatCatalog No.

Price ($US)

DNMT Activity / Inhibition Assay

1 x 96 rxns

55006

495

Recombinant DNMT1 protein, active

10 µg

31335

320

NEW: Dounce Homogenizer to Assist in Cell and Chromatin Preparations
Obtain more complete cell lysis
Whether you are analyzing cell lysatesby Western blot, or preparing chromatin
for a chromatin immunoprecipitation
experiment, the ability to obtain goodresults relies on accurate cell lysis. For
cell and tissue samples that seem to beresistant to lysis under normal detergent
conditions, the use of a dounce homogenizer can greatly improve lysis, thereby
improving the recovery yields of protein
or chromatin material.
Active Motif’s new Dounce Homogenizer is ideal for preparation of cell lysates

8

North America 877 222 9543

or chromatin. The dounce is available intwo sizes, 1 ml for small sample volumes
and 15 ml for larger sample preparations.
Each glass dounce is supplied with two
pestles of different sizes. One pestle ﬁts
tightly within the shaft of the dounce
for maximum friction and cell disruption.
The other pestle has a looser ﬁt and isperfect to obtain a homogenous sample.
Because the dounce homogenizer ismade of glass, it can easily be cleaned
and sterilized between uses.

Figure 1: Dounce homogenizer with two pestles.

Product

FormatCatalog No.

Price ($US)

Dounce Homogenizer

1 ml
15 ml

40401
40415

175
225

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Japan +81 (0)3 5225 3638

www.activemotif.com

October 2011 • volume 12 • number 2

Sample Preparation

NEW: Protease Inhibitor Cocktail for Better Sample Preparation
Broad-spectrum Protease Inhibitors
The same Protease Inhibitor Cocktail you
have trusted in Active Motif’s Nuclear
Extract Kit (see details below) is now
available separately. The cocktail is designed to prevent proteolytic degradation during lysis and extraction procedures from mammalian cells or tissues.
The Protease Inhibitor Cocktail contains
a mixture of several proteases to offera broad-spectrum of protection. The
cocktail has inhibitors targeting cysteine,
serine and aspartic acid proteases, aswell as inhibitors for aminopeptidases.
In addition to being ready-to-use, theProtease Inhibitor Cocktail is also
supplied EDTA-free. The cocktail is packaged as a 100X liquid solution that has
been stabilized in DMSO for improved
accuracy, solubility and ease of use incomparison to traditional tablets. Selectfrom either 1 ml (2 x 500 µl) or 5 ml packsizes to suit your research needs.

Protease Inhibitor Cocktail advantages• Broad-spectrum inhibition• Optimized for use with mammaliancell and tissue extraction• Convenient and stable liquid
format for ease of use• Supplied as a ready-to-use
100X stock that is EDTA-free

To see the complete Protease Inhibitor Cocktail
formulation, visit our website at www.activemotif.com/pic.
Product

FormatCatalog No.

Price ($US)

Protease Inhibitor Cocktail

1 ml
5 ml

37490
37491

95
295

Nuclear Extraction Kit for Nuclear, Cytoplasmic or Whole-cell Lysates
Active Motif’s Nuclear Extract Kit is ideal for researchers interested in isolating high-quality nuclear, cytoplasmic orwhole-cell lysates from mammalian cell or tissue samples. The optimized protocols and reagents simplify the
extraction procedure so you obtain high yields every time.
How does the Nuclear Extract Kit generate such highly specific cell fractions?
Each Nuclear Extract Kit provides
reagents for 100 or 400 extractions from
8.8 x 106 cells, which corresponds to
HeLa cells grown to conﬂuence in a
100 mm tissue culture dish. First, thecells are collected in ice-cold PBS in
the presence of Phosphatase Inhibitors
to limit further protein modiﬁcations,such as proteolysis, dephosphorylation,etc. Then, the cells are resuspended
in Hypotonic Buffer to swell the cell
membrane and make it fragile. The
addition of detergent causes leakageof the cytoplasmic proteins into thesupernatant, where they can be collected for further analysis. The remaining
nuclear pellet is lysed and the nuclear
proteins are solubilized in Lysis Buffer
supplemented with our broad-spectrum
Protease Inhibitor Cocktail.

www.activemotif.com

Figure 1: Specific extraction of cytoplasmic andnuclear extracts as tested in TransAM® NFκB p50 Kit.
Nuclear and cytoplasmic extracts were prepared using
the Nuclear Extract Kit from HeLa cells that were
unstimulated or stimulated with TNF-α for 30 minutes.
The extracts were assayed using TransAM NFκB p50
at the concentrations shown. Because activated NFκB
translocates to the nucleus, only nuclear extracts fromstimulated cells should contain activated NFκB.

For whole-cell lysate preparation, thecells are collected in PBS containing
Phosphatase Inhibitors and then lysedin the Lysis Buffer containing Protease
Inhibitor Cocktail.
Following protein quantiﬁcation, the cytoplasmic, nuclear or whole-cell extracts

can be used in a variety of downstream
applications, including Western blotting,
EMSA, transcription factor-DNA binding
assays, such as Active Motif’s TransAM®
Assay Kits, or they are ready for use in
Active Motif’s DNMT Activity / Inhibition Assay (see page 8).

Product

FormatCatalog No.

Price ($US)

Nuclear Extract Kit

100 rxns
400 rxns

40010
40410

205
585

Japan +81 (0)3 5225 3638

Europe +32 (0)2 653 0001

North America 877 222 9543

9

Stem Cell & Nuclear Receptor Antibodies

Antibodies to Stem Cell Regulators to Further your Stem Cell Research
The biology of stem cells involves a wide variety of proteins found in
the nucleus that are required to maintain cell pluripotency (the ability
to differentiate into any cell type) and self-renewal. Many of these
proteins are associated with chromatin and serve to keep large regionsof the genome in an open, accessible conﬁguration. In fact, chromatin
biology and epigenetics are integral components of stem cell biology.
To assist you in your study of stem cells and the role of chromatin
regulation, Active Motif offers a wide range of antibodies toimportant stem cell proteins, including:• Transcription factors

Figure 1: Ring1B staining in U2OS cells.
Human U2OS cells were stained with Ring1B monoclonal
antibody (Catalog No. 39663) and visualized by indirect
immunofluorescence.

• Chromatin modifying proteins• Histones and histone modiﬁcations• Polycomb proteins
For more information on our antibodies to regulators of stem cell function, visit www.activemotif.com/stemcellabs.

Highly Validated Antibodies and ELISAs for the Study of Nuclear Receptors
Nuclear receptors are important regulators of endocrine activity, mediating
responses to changing hormone levels.
Most nuclear receptors bind steroid
hormone ligands. But, there are some(orphan receptors) that do not, or forwhich the ligand has not yet been identiﬁed. Because nuclear receptors can bind
DNA directly and regulate gene expression, they are also transcription factors,
many of which recruit co-activatorsand co-repressors that possess histonemodifying activity. Active Motif offers
a number of highly validated antibodies
for the study of nuclear receptors andsteroid hormone activity.

Figure 1: Immunoﬂuorescence staining ofProgesterone receptor.
Mouse hypothalamic section stained with Progesterone
receptor antibody (Catalog No. 61023).

Figure 2: Androgen receptor analyzed by chromatin IP.
ChIP was performed using 30 µg of VCAP60 cell chromatin and 10 µl of Androgen receptor antibody (Catalog
No. 39781). ChIP DNA was used in qPCR with the Human
Negative Control Primer Set 1 (Catalog No. 71001) andprimer pairs specific for the KLK3 and FKBP5 genes. Data
are presented as Binding Events Detected per 1000 Cells.

For more information on Active Motif’s
antibodies to nuclear receptors, visit
www.activemotif.com/nrabs.

Active Motif also offers sandwich ELISAs
that enable you to quantitatively measure the levels of the Androgen, Estrogenand Progesterone nuclear receptors. For
more complete information, please visit
www.activemotif.com/nrelisa.

10North America 877 222 9543

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www.activemotif.com

October 2011 • volume 12 • number 2High Resolution Microscopy

Antibodies and Reagents for High Resolution Confocal STED Microscopy
The resolution attainable in ﬂuorescent microscopy is limited by the diffraction of light, which was shown by Ernst
Abbe in 1873. The Abbe Limit restricts the ability of the observer to visually resolve objects that are separated byless than ~200 nm. Recently, several super-resolution techniques have been developed to overcome this limitation,
providing the ability to image structures as small as 20 nm. These improvements in microscopy enable scientists todecipher the nanostructure of the cell in details that could not be visualized before.
One of these novel techniques is STED
(STimulated Emission Depletion), which
can achieve up to 12-fold higher resolution than classical confocal microscopy.
Active Motif is proud to help scientists
take advantage of this powerful technology by providing IF-tested primary antibodies, and high-quality conjugated secondaries & sample preparation reagentsthat are certiﬁed by Leica Microsystems
for use with its STED microscopes. For
more complete information on STED,
please visit www.activemotif.com/sted.
Optimized sample preparation for
STED and classical confocal microscopy
Proper sample preparation is among themost signiﬁcant factors for obtaining
high-quality images in both STED andclassical confocal microscopy. To help
ensure that you consistently achieve
the best results possible, Active Motif’sscientists developed the Chromeo™ STED

Immunoﬂuorescence System and the
MAX Stain™ Immunoﬂuorescence Tools.
These sample preparation reagents were
designed to increase the intensity andspeciﬁcity of your ﬂuorescent labeling.
Customized conjugation protocols
ensure the highest quality secondaries
The quality of any ﬂuorescent secondary antibody depends on the spectral
properties of the dye, the quality of thesecondary antibody, and the dye-toprotein ratio & purity of the conjugate.
To control all variables possible, Active
Motif produces all its conjugates using
protocols that have been optimized foreach dye/antibody combination. This
ensures the quality of the conjugates,
and makes the ﬂuorescent secondaries
brighter while lowering the ﬂuorescentbackground. For more information, visit
www.activemotif.com/secondary.

Product

FormatCatalog No.

Price ($US)

Chromeo™ 488 Goat anti-Mouse IgG

1 mg

15031

135

Chromeo™ 488 Goat anti-Rabbit IgG

1 mg

15041

135

Chromeo™ 505 Goat anti-Mouse IgG

1 mg

15030

135

Chromeo™ 505 Goat anti-Rabbit IgG

1 mg

15040

135

Chromeo™ 494 Goat anti-Mouse IgG

1 mg

15032

135

Chromeo™ 494 Goat anti-Rabbit IgG

1 mg

15042

135

ATTO 647N (STED) Goat anti-Mouse IgG

250 µl15038

260

ATTO 647N (STED) Goat anti-Rabbit IgG

250 µl15048

260

ATTO 655 (STED) Goat anti-Mouse IgG

250 µl15039

260

ATTO 655 (STED) Goat anti-Rabbit IgG

250 µl15049

260

Chromeo™ STED Immunoﬂuorescence System

1 kit

15260

230

MAXpack™ Immunostaining Media Kit

1 kit

15251

295

www.activemotif.com

Japan +81 (0)3 5225 3638

Europe +32 (0)2 653 0001

Figure 1 Active Motif’s primary and ﬂuorescentsecondary antibodies in CW STED microscopy.
HeLa cells were stained with alpha Tubulin mouse mAb(Clone 5-B-1-2) (Catalog No. 39527) and Chromeo 505
Goat anti-mouse IgG (Catalog No. 15030). The confocal
image (top) and the STED image (bottom) are courtesyof Leica Microsystems, Germany.

Figure 2: Active Motif’s primary and ﬂuorescentsecondary antibodies in TCS STED microscopy.
HeLa cells were stained with alpha Tubulin mouse mAb(Clone 5-B-1-2) (Cat. No. 39527) and Chromeo 494 Goat
anti-mouse IgG (Cat. No. 15032). Histone H3 was stainedwith Histone H3 trimethyl Lys4 rabbit polyclonal antibody (Cat. No. 39159) and the ATTO 647N (STED) Goat
anti-rabbit IgG (Cat. No. 15048) secondary antibody. The
STED image is courtesy of Leica Microsystems, Germany.

North America 877 222 9543

11

Enabling Epigenetics Research

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, MeDIP, 5-hmC & 5-mC antibodies

Visit us at www.activemotif.com

Superior Antibodies & Kits for

EPIGENETICS RESEARCH