IN THIS ISSUE

Reagents for Gene Regulation & Epigenetic Research
2 NEW: Study Gene Regulation with theLightSwitch™ Luciferase Assay System

9 ChIP Products Overview

4 Antibody Use in Drug Discovery Research

10 NEW: Histone PTM Multiplex Assays for
Use on Luminex® Instruments

5 Expanding Our Offering of New,
Cutting-edge Epigenetic Assays

12 NEW: Screen Protein Domains for
Histone Binding Partners

6 NEW: Perform ChIP on FFPE Samples
with the ChIP-IT® FFPE Kit

14 MethylCollector™ Ultra for the Most Sensitive
Enrichment of Methylated DNA

8 NEW: Ready-to-use ChIP Columns andFixation Buffer for Your ChIP Protocols

15 NEW: CDy1 Fluorescent Dye for Identiﬁcation
and Imaging of Live Stem Cells

Luciferase Reporter Constructs and Assays

NEW: Study Gene Regulation with the LightSwitch™ Luciferase Assay System
Active Motif’s LightSwitch™ Luciferase Assay System is a complete solution for performing gene regulation studies
in living mammalian cells, as well as for functional validation of protein binding events detected in ChIP assays. With
GoClone™ Collections of over 18,000 human promoters and 12,000 3´UTRs available as transfection-ready LightSwitch
luciferase reporter vectors, you can perform your reporter assay experiments immediately, without the need toclone or prepare DNA. Optimized assay & transfection reagents and protocols have been developed for every step of
the process, making the entire procedure faster and simpler than with other methods. Also available are positive andnegative control vectors, stable cell lines, miRNA mimics & inhibitors, and a collection of cloned long-range synthetic
response elements. And, our services team can provide custom cloning, mutagenesis, pathway screening andsequence variant analysis. For more complete information, please visit www.activemotif.com/lightswitch.
LightSwitch GoClone Promoter and3´UTR Reporter Collections
The LightSwitch GoClone Collections
make it fast and easy for you to measure
the transcriptional and post-transcriptional regulation of nearly any gene in
the genome. To begin, simply searchfor your elements of interest using ouronline search tool (Figure 1), then you’reready to begin your gene regulation
studies immediately.

LightSwitch 3´UTR Reporter Constructs
are created by cloning a human 3´UTRdownstream of the RenSP gene in thepLightSwitch_3UTR plasmid. The vector’s constitutive RPL10 promoter drives
expression of hybrid luciferase-3´UTRtranscripts. Assays are performed todetermine how the miRNA-3´UTRinteractions affect luciferase activity.

2

In the box below, paste in a list of one or more gene identiﬁers on
separate lines. Click on one of the buttons below to search.
DNMT3ADNMT3B

Search for:

Prod ID

Promoter

3´UTRGene Symbol

Both

Type

Sequence of Cloned Elements

Format

S709833

DNMT3APROMOTER

Sequence and Clone Info

5 µg

S709808

DNMT3B

PROMOTER

Sequence and Clone Info

5 µg

S808608

DNMT3A

3´UTRSequence and Clone Info

5 µg

S809202

DNMT3B

3´UTRSequence and Clone Info

5 µg

Figure 1: Online search tool for ﬁnding LightSwitch Promoter and 3´UTR reporter vectors.
Finding cloned regulatory elements for your genes of interest is fast and easy. Simply enter your gene’s name, accession
number or other identifier in the search box and click a button to search. The search results detail which GoClones
constructs are available for your elements, including links to view gene information at NCBI and the cloned sequence.

5´

Bgl I
Sac I
Mlu I
Nhe I
Xho I
Bgl II
Hind IIIThe backbone of the LightSwitch Systemis the LightSwitch Promoter and 3´UTRreporter vectors (Figure 2). LightSwitch
Promoter Reporter Constructs are created by isolating a promoter from thehuman genome and cloning it into themultiple cloning site that is upstreamof the RenSP luciferase reporter geneon the pLightSwitch_Prom plasmid. The
promoter activity can then be measured
simply by assaying for the luminescencesignal (Figures 3 & 4).

SEARCH BY GENE IDENTIFIER

Ampr

Constitutive
promoter

3´

RenSP

pLightSwitch_Prom
3656 bp

Ampr

RenSP

pLightSwitch_3UTR
3910 bp

poly Aori

5´

poly A
ori

Xba I
Nhe I
Avr II
Xho I
Fse I
3´

Figure 2: Maps of empty LightSwitch Promoter and 3´UTR reporter vectors.

Toll Free — 1 877 222 9543

June 2013 • volume 14 • number 2
Optimized luciferase and assay
reagents for the best results possible
All LightSwitch vectors utilize RenSP,
an optimized Renilla luciferase genethat has been engineered for maximum
brightness and minimal background.
Starting with the native Renilla gene,
thousands of synthetic gene sequencevariants were functionally screened forincreased enzymatic activity (light output). A protein destabilization domain
was added to decrease the half-life of
the RenSP protein. And, transcription
factor binding sites were removed asthese might confound expression measurements. In addition, the LightSwitch
Assay Kit features a novel, proprietary
substrate and an optimized lysis bufferthat were formulated to provide highsensitivity over a broad dynamic range
when used with our engineered RenSP.
Human genome
Promoter

Gene of interest

Cloned into
Reporter
VectorRenSP
Luciferase

Luciferase Reporter Constructs and Assays

Transcriptional vs. Post-transcriptional Regulation
Studying transcription factor activity with promoter reporter assays
Transcription factors interact with promoters to modulate when, where and how
a gene’s mRNA is made. Transcription factor activity can be affected by the cell’senvironment including exposure to signaling molecules like hormones and growth
factors. With the LightSwitch System, researchers can monitor changes in any
promoter’s activity in response to variations in transcription factor function that
may be modulated by techniques like over-expression, knock-down or treatment
with a known agonist or antagonist. LightSwitch Promoter Reporter constructs arealso useful for assessing the functional consequences of transcription factor binding
detected through ChIP experiments, as well as for sequence variant assays thatmeasure the effects of mutagenizing a transcription factor’s binding motif.
Post-transcriptional regulation through miRNA-3´UTR interactions
Gene regulation studies have historically focused on events that inﬂuence transcription, such as the binding of transcription factors to promoters. More recently, muchresearch is centered on the presence or absence of histone modiﬁcations that inﬂuence DNA accessibility by altering chromatin structure. However, post-transcriptional
regulation plays an equally important role. The interaction of microRNAs (miRNAs)
with the 3´UTRs on transcribed mRNAs affect both a transcript’s stability and its
rate of translation. Interestingly, miRNAs seem to play a role in regulating proteins
involved in regulation of chromatin structure (e.g. EZH2, HDAC1 and DNAMT3A & 3B).
For miRNA-3´UTR interaction studies, utilize our 12,000 LightSwitch 3´UTR Reporter
constructs, 900 synthetic miRNA target constructs, and 1000 miRNA mimics andinhibitors, which are for co-transfection with the 3´UTR & miRNA target constructs.

LightSwitch™
Promoter
ReporterComplete solution for gene regulation
LightSwitch is the most comprehensive
set of reagents for the study of gene
regulation. For details, please visit us atwww.activemotif.com/lightswitch:

Transfect
VectorLight
SignalPromoter

Luciferase

Figure 3: The LightSwitch Luciferase Assay work ﬂow.
After selecting a pre-cloned LightSwitch Promoter(shown above) or 3´UTR Luciferase Reporter Vector,
it is transfected into an appropriate cell line. The cells
are stimulated, if required, to induce transcription of
RenSP luciferase. Because RenSP catalyzes oxidation ofits substrate in a reaction that produces light, promoter
activity is quantified using a LightSwitch Assay Kit and aluminometer to measure the luminescence signal.

www.activemotif.com

Figure 4: Response of LightSwitch SYT8 Promoter
Reporter vector to various estrogen compounds.
Dose response data is shown for the SYT8 promoter(Product ID S714388) for five different estrogenic compounds. The graph summarizes the luciferase activityof this promoter in HT1080 human fibrosarcoma cells
that were co-transfected with an ER cDNA expression
plasmid, then treated with various estrogen compounds
for 24 hours before the luminescence was measured
using the LightSwitch Luciferase Assay Kit. The Y-axis
shows relative luminescent values; the X-axis shows themolar concentration of compound on a log10 scale. The
Coefficients of Variance (CVs) ranged from 10% to 12%• 18,000 human promoter and12,000 human 3´UTR constructs• Positive and negative controls• Synthetic TF response elements• Empty Vector Suite• FuGene® HD & DharmaFECT® Duo
Transfection Reagents• Synthetic miRNA target constructs• miRNA mimics and inhibitors• Stable cell lines• LightSwitch™ Luciferase Assay Kit• Custom services

3

Drug Development Assays

Antibody Use in Drug Discovery Research
Epigenetic targets are the most promising class of drugable targets to emerge in a decade as they are not only
relevant to oncology research, but also have potential in metabolic, neurological, inﬂammatory and cardiovasculardisorders. Antibodies are an important tool used in drug development to better understand the epigenetic changes
and potential off-target effects. Active Motif’s extensive portfolio of antibodies against DNA methylation, histone
modiﬁcations and histone modifying enzymes will help advance research in all phases of drug discovery.
TargetValidation

Target ID

Minimize risks
Active Motif specializes in manufacturing antibodies against histone modiﬁcations and chromatin proteins. Because
we manufacture and test our own antibodies, we can deliver the high-quality
antibodies that your research requires.

PreclinicalDevelopment

ScreenTools to build assays
Active Motif antibodies are compatible with many applications and can be combinedwith our recombinant modiﬁed histones and recombinant histone modifying enzymes
to build assays quickly. Antibodies against modiﬁed histone and histone modifying
enzyme pairs include: EZH2 & H3K27me3; MMSET & H3K36me2; LSD1 & H3K4me2;
DOT1L & H3K79me2.
337 nmHistone H3K4me2 polyclonal antibody(Catalog No. 39141)

Assay compatible formulations
Antibody formulations containing glycerol, BSA, amine-containing azides, Tris or
glycine can wreak havoc on establishedassays. Active Motif offers antibodies
in PBS, so that they can be seamlessly
integrated into existing pipelines.
Large lot sizes
We produce large lots to ensure consistency in all phases of the drug discovery
process, including antibody-based HTS.

K79me3

K79me2

K79me1

K79

K9me3

K9me2

K9me1

K9

K4me3

K4me2

K4me1

(CH3)2(Catalog No. 31277)(CH3)2LSD1
H3665 nmH3
H3

Figure 2: Time-Resolved FRET assay to monitor demethylation of H3K4 by LSD1.

DRUG TARGET ANTIBODIES
Target
HMTs

HDMs

Figure 1: Dot blot analysis of Histone H3K36me3 pAb.
The reactivity of Histone H3K36me3 pAb (Cat. No. 61101)
to various H3 lysine modifications was tested by dot blot.

4

Recombinant Histone H3K4me2 protein

HATs

K36me3

K36me2

K36me1

K36

K27me3

K27me2

K27me1

K27

K23me3

K23me2

K23me150
10(Catalog No. 31334)

HDACs
K23

pg of peptide

50
10

pg of peptide

K4

Speciﬁcity is rigorously tested
Active Motif histone antibodies aretested for cross-reactivity to multiple
histone tail modiﬁcations.

590 nmRecombinant LSD1 protein

Readers
DNMTs

Antibody
DOT1L antibody (pAb)
EZH2 antibody (pAb)
MLL / HRX antibody (pAb)
JARID1C / KDM5C antibody (pAb)
JMJD2A antibody (mAb)
JMJD2B / KDM4B antibody (pAb)
GCN5 antibody (mAb)
MOF / MYST1 antibody (pAb)
HDAC1 antibody (pAb)
HDAC2 antibody (mAb)
SIRT1 antibody (mAb)
BRD4 antibody (pAb)
PELP1 antibody (pAb)
DNMT1 antibody (mAb)
DNMT2 antibody (pAb)
DNMT3A antibody (mAb)

Format100 µl100 µl100 µg200 µl100 µg100 µl100 µg100 µl100 µg200 µl200 µg100 µl100 µl100 µg100 µg100 µgCatalog No.
39953
39901
61295
39229
39815
61221
39975
61245
40967
39533
39353
39909
61263
39204
39205
39206

Toll Free — 1 877 222 9543

June 2013 • volume 14 • number 2

Products for Chromatin Immunoprecipitation

Expanding Our Offering of New, Cutting-edge Epigenetic Assays
Active Motif’s Epigenetic Services is world-renowned as the leading provider of end-to-end ChIP-Seq services. With
over 10 years of experience providing ChIP services, our team has the experience and know-how that allows us topush the envelope and offer the latest technological advances in ChIP and MeDIP.

NEW: Active Motif will perform ChIP-Seq from your FFPE samples!
Breakthroughs at Active Motif’s Epigenetic Services lab have resulted in technology that enables us to generate
genome-wide proﬁles of histone modiﬁcations using formalin-ﬁxed, parafﬁn-embedded (FFPE) samples.
FFPE tissue blocks and histology slides
are a valuable resource for retrospective
research on clinical samples. Clinical information, treatments and outcomes areoften available for these sample types.
This provides researchers with an opportunity to link data from FFPE samples todisease, diagnosis and biomarker discovery. In the ﬁeld of epigenetics, until now,the beneﬁts of this archived material
have not been fully realized due to thelimited size of the samples and the lossof antigenicity due to the harsh conditions used to prepare these samples.

DehcWb
9ebed

9ebed
Jkceh

Figure 1: H3K4me3 ChIP-Seq on normal and tumor
human colon FFPE samples.
Chromatin was extracted from 10-year-old histologicalsections of a colon tumor and matched normal colon.
The chromatin was used in H3K4me3 ChIP-Seq to generate genome-wide profiles of this histone modification.
The expected promoter enrichment at > 12,000 genes
was detected. A portion of that data is presented in the
figure. It shows nearly equal H3K4me3 occupancy at the
SRF and CUL9 promoters. However, H3K4me3 is present
at the promoter of the PTK7 gene, a gene known to be
up-regulated in colon cancer, only in the tumor sample.

NEW: Genome-wide proﬁles of all cytosine variants now available
Active Motif’s Epigenetic Services team has expanded our DNA analysis portfolio to include genome-widehydroxymethylcytosine (hmC), formylcytosine (fC) and carboxylcytosine (caC) proﬁling. Simply send us yourDNA, cells or tissues and we will deliver the data.
Advances in DNA methylation research
have led to the discovery of multiple
cytosine variants that are derived fromthe oxidation of 5-methylcytosine
through the activity of the Tet family of
enzymes. These modiﬁcations include
5-hmC, 5-fC and 5-caC. Given that these
are relatively recent discoveries, the biological relevance of these modiﬁcations
is not fully understood. Are these simply
by-products along the way to DNA
demethylation? Do they mark speciﬁcregions of the genome? Do they recruitother important proteins? Let us help
you be the ﬁrst to know!

www.activemotif.com^c9

Figure 2: Genome-wide proﬁling of DNA variants.
DNA was extracted from a human tumor and enrichment was performed using antibodies against hmC, fC
and caC. The resulting profiles identified over 100,000
genomic locations marked by these DNA variants. The
figure shows one gene, TUSC5, that is marked in the
promoter and across the gene body by all three variants.
The intensity of the peaks reflects the expected abundance of each DNA variant.

\9

YW9

To obtain information on
all of our Epigenetic Services,
please contact us atservices@activemotif.com.

5Chromatin Immunoprecipitation

NEW: Perform ChIP on FFPE Samples with the ChIP-IT® FFPE Kit
Active Motif has a long history of innovation in chromatin immunoprecipitation assays. We develop technologies andutilize quality reagents to make the fastest, easiest, most sensitive ChIP kits available. Because we are able to achieve
such a high level of sensitivity, Active Motif has been able to develop a new ChIP-IT® FFPE Chromatin Preparation Kit
and a new ChIP-IT® FFPE Kit. These ﬁrst-of-their-kind products enable extraction of chromatin from FFPE slides ortissue blocks for use in ChIP analysis.

First of its kind assay
Formalin-ﬁxed parafﬁn-embedded (FFPE)
tissue serves as the gold standard forthe preservation of pathology samples.
As a result, large collections of FFPE
samples are commercially available.
These samples have the potential toprovide valuable information about theepigenetic variances between normal
and diseased tissue states.
Traditionally, FFPE samples have not been
useful in chromatin immunoprecipitation
because of the difﬁculty in obtaining
high-quality chromatin from slides or tissue blocks. The formalin ﬁxation processoften causes degradation and loss ofantigenicity. With Active Motif’s new
ChIP-IT® FFPE Chromatin Preparation Kit,
high-quality chromatin can be extracted
from FFPE samples and, when used in
combination with the ChIP-IT FFPE Kit,
the assay is sensitive enough to obtain
ChIP-enriched DNA for analysis by qPCR
or Next-Generation sequencing.
What’s in the box?
The ChIP-IT FFPE Chromatin Preparation Kit provides specially formulated
reagents and protocol guidelines to extract chromatin from preserved samples,
including human FFPE tissues. While thequality of the extracted chromatin isdependent upon sample ﬁxation andstorage conditions, we have successfully
extracted ChIP-grade chromatin from6

normal and tumor human colon FFPE
blocks that were stored for more than
10 years under less than ideal conditions(Figure 1).

Figure 1: FFPE tissue blocks of normal and tumor
samples from human colon stored for over 10 years.
Images of the normal and tumor human colon FFPE
blocks used with the ChIP-IT FFPE Chromatin Preparation
Kit. These samples had been stored in an uncontrolledenvironment for more than 10 years. Five 20 µm sections
from each sample were pooled and processed to obtainenough chromatin for use in the ChIP-IT FFPE Kit.

Important considerations
Optimization of extraction conditions
will depend on the size and tissue type.
Our detailed protocol offers guidelines
and troubleshooting tips for processing
samples to obtain sufﬁcient chromatin
for use in downstream ChIP analysis. The
kit also includes positive control DNA
and primers to help validate chromatin
quality by qPCR analysis prior to proceeding with the ChIP reactions. Table 1
lists examples of the sample material
we have successfully validated with the
ChIP-IT FFPE Chromatin Preparation Kit.

FFPE sample
Human Colon

Sample used per chromatin prep
Tissue block – ﬁve 20 µm sections

Human Kidney

Tissue block – twenty-ﬁve 20 µm sections

Human Lung

Tissue block – two 20 µm sections

Rat whole brain

5 slides – two 5 µm sections per slide

Rat hippocampus

25 slides – two 5 µm sections per slide

Table 1: Examples of FFPE samples successfully used with the ChIP-IT FFPE Kit.

Toll Free — 1 877 222 9543

June 2013 • volume 14 • number 2

),''
)'''
(,''
('''
,''
'

www.activemotif.com()''?ldXeE\^Gi`d\iJ\k)?ldXeGfjGi`d\iJ\k>8G;?$)('''

9^?Fki_d]OO'7dj_XeZo
EfidXc
Kldfi

/''
-''
+''
)''
'

?ldXeE\^Gi`d\iJ\k(?ldXeGfjGi`d\iJ\k8K>+9

Figure 2: qPCR analysis of normal and tumor human colon samples assayed using ChIP-IT FFPE.
The normal and tumor human colon samples shown in Figure 1 were sliced into 20 µm sections; five sections per sample
were pooled and used to prepare one chromatin preparation each with the ChIP-IT FFPE Chromatin Preparation Kit. The
chromatin quality was validated using the included positive control DNA and primer sets. 300 ng and 200 ng of normal andtumor chromatin, respectively, were used per ChIP reaction using the ChIP-IT FFPE Kit. Antibodies for histone H3K4me3 ortranscription factor YY1 were used for enrichment according to the recommendations in the manual. The quality of the
ChIP-enriched DNA was then validated using the ChIP-IT qPCR Analysis Kit, which enables normalization of the data to
account for differences in chromatin amounts, primer efficiency and ChIP elution volumes. The qPCR results for each ChIP
antibody are shown above. The H3K4me3 results match observed data in which GAPDH is upregulated in certain cancers.

ChIP-IT FFPE advantages• Obtain quality chromatin – use
slides or tissue blocks as input• Sample versatility – works withhuman, mouse or rat samples• Positive controls – included inboth the chromatin preparation
and ChIP kits to validate results
at each step of the process• Validated – ChIP-enriched DNA
was tested using both qPCR &ChIP-Seq to conﬁrm theenrichment of histone andtranscription factor targets

For more complete details, or to
download a copy of our
product manuals, please visit
www.activemotif.com/ffpechip.

9^?Fki_d]>)A*c[)7dj_XeZo
EfidXcKldfi
9`e[`e^\m\ekj[\k\Zk\[&('''Z\ccj*'''9`e[`e^\m\ekj[\k\Zk\[&('''Z\ccj

Enrich for histone or transcription
factor targets with the ChIP-IT FFPE Kit
To perform ChIP analysis on the chromatin extracted from FFPE samples it isnecessary to use the new ChIP-IT FFPE
Kit. This is the only ChIP Kit available
that has the sensitivity required to workwith extremely limited starting material
while producing minimal backgroundsignal, thereby enabling speciﬁc detection of the target protein of interest.
Alternative ChIP kits are unable toprovide the level of sensitivity required
for speciﬁc enrichment. The ChIP-IT
FFPE Kit includes a positive control
antibody to validate successful ChIP
reactions. For downstream applications
such as Next-Generation sequencing,
the ChIP-IT qPCR Analysis Kit is recommended to evaluate the quality of the
ChIP-enriched DNA.

Chromatin Immunoprecipitation

EfidXc:fcfe

PP(:_@G

EfidXc:fcfe?*B+d\*:_@G

Kldfi
:fcfe

?*B+d\*:_@G

Figure 3: ChIP-IT FFPE ChIP-Seq results using normal and tumor human colon samples.
H3K4me3 ChIP-Seq results from tumor and normal FFPE colon samples and YY1 ChIP-Seq fromnormal colon are displayed above. Only the portion of the genome-wide data surrounding the YY1gene is shown. The top panel shows YY1 binding at the promoter of the YY1 gene, thus illustrating
the known autoregulation of YY1. The middle and bottom sequence represent H3K4me3 binding at
the YY1 gene in normal and tumor, respectively, indicating YY1 is expressed in both the normal andtumor samples.

Product

FormatCatalog No.

ChIP-IT® FFPE Chromatin Preparation Kit

5 rxns

53030

ChIP-IT® FFPE

16 rxns

53045

ChIP-IT® qPCR Analysis Kit

10 rxns53029

7

Chromatin Immunoprecipitation

NEW: Ready-to-use ChIP Columns and Fixation Buffer for Your ChIP Protocols
For labs that routinely perform ChIP, Active Motif is making it faster and easier to obtain consistency in chromatin
preparations and improve the quality of the ChIP reactions. The addition of ChIP-IT® Fixation Buffer during chromatin extraction improves both the consistency of sample preparation and also enhances ChIP efﬁciency. With Active
Motif’s ready-to-use Protein G Agarose Columns, simply add the ChIP reaction directly into the column to perform
incubation and washing steps. The columns provide a faster alternative to centrifugation methods and ensurecomplete sample recovery for better reproducibility.
Ready-to-use ChIP Columns
Active Motif’s new Protein G Agarose
Columns provide the perfect solution
for labs that routinely perform ChIP. The
columns contain high-afﬁnity protein
G agarose beads that strongly bind IgG.
The beads have been speciﬁcally engineered to eliminate non-speciﬁc binding,making them a better option than traditional agarose or protein G magnetic
beads. The protein G agarose supplied ineach column has a binding capacity of 10µg IgG/µl bead, and can be adapted towork with any ChIP protocol.
The beads have been pre-washed andloaded into ﬁltration columns. Simply
add the ChIP reaction to the columns
and use the cap to seal. The incubation
and wash steps are all performed within
the column to streamline the process
and prevent the loss of any material.
This provides a faster solution tocentrifugation and magnetic separation
methods, and results in better reproducibility between samples because nomaterial is lost.
In addition to ChIP, the Protein G
Agarose Columns can also be used for
co-immunoprecipitation (Co-IP) experiments. The columns have been validated
using Active Motif’s Nuclear Complex
Co-IP Kit (Catalog No. 54001), where
the strong binding afﬁnity and reduced
background of the protein G agarose
make these columns an ideal tool for
antibody capture during Co-IP.

8

Figure 1: Image of a Protein G Agarose Column.
ChIP reactions can be added directly to the Protein G Agarose Column andincubations are performed directly in the column. To wash, simply snap off
the tab at the bottom and place the column in an empty 1 ml pipet tip box
for use as a holder to perform the wash steps.

ChIP-IT Fixation Buffer
The unique properties of the ChIP-IT
Fixation Buffer provide consistency between chromatin preparations for better
reproducibility across experiments. This
specially formulated buffer is combinedwith formaldehyde and added directly
to the cell culture growth medium (withor without serum) or to tissue material.
The buffer helps eliminate pH effects
during chromatin preparation for moreconsistent ChIP results.

Protein G Column advantages• Ready-to-use – the beads havebeen pre-washed and loaded
into the column• No pre-blocking needed – theprotein G agarose beads havebeen engineered to eliminate
non-speciﬁc binding• Avoid sample loss – the column
ensures complete retention of
the beads to prevent sample loss
• Versatility – use them for both
ChIP and Co-IP experiments

Product

FormatCatalog No.

ChIP-IT® Fixation Buffer

3 ml

53038

Protein G Agarose Columns

30 rxns

53039

Toll Free — 1 877 222 9543

June 2013 • volume 14 • number 2

Products for Chromatin Immunoprecipitation

ChIP Products Overview
Active Motif has consistently introduced innovations that make ChIP faster, simpler and more reproducible. We werethe ﬁrst to introduce magnetic beads into a ChIP kit, which lead to the ﬁrst kits for sequential ChIP, HT and RNA ChIP.
Our latest kits make it possible to perform ChIP with low-binding afﬁnity antibodies and from FFPE samples (page 6).
We also offer analysis products to validate your ChIP results, including 18,000 cloned promoters for functional validation of protein binding (page 2). The diagram below highlights some of our products for each step of the ChIP process.
Chromatin Preparation

Chromatin Shearing• EpiShear™ Sonicators• ChIP-IT® Sonication &Enzymatic Shearing Kits
• Dounce Homogenizers• FFPE Chromatin Prep Kit

Pre-Made Chromatin• Ready-to-ChIP Chromatin

ChIP Antibodies

Chromatin Immunoprecipitation• ChIP & ChIP-Seq
validated antibodies• ChIP Antibody
Validation Services• MODiﬁed™ Histone
Peptide ArrayChIP Kits• ChIP-IT® Express• ChIP-IT® High Sensitivity• ChIP-IT® ChIP-Seq• Re-ChIP-IT®
• RNA ChIP-IT®
• ChIP-IT® FFPEDNA Clean-up• Chromatin IP
DNA Puriﬁcation Kit

ΔRxn

Downstream Analysis

Cycle

PCR Analysis• ChIP-IT® Control Kits

Luciferase Reporter Assays• LightSwitch™ Promoter Reporter
GoClone™ Collections• LightSwitch™ Stable Cell Lines• LightSwitch™ Luciferase
Assay Reagents

www.activemotif.com

qPCR Analysis

Epigenetic Services• ChIP-IT® qPCR Analysis Kit• ChIP-IT® qPCR Control Kits
• ChIP Control qPCR Primer Sets

Functional Validation
Fhecej[hGene Regulation Services

BkY_\[hWi[Fhecej[hH[fehj[hL[Yjeh• FactorPath™ ChIP-Seq & ChIP-chip• TranscriptionPath™ ChIP-Seq & ChIP-chip• HistonePath™ ChIP-Seq & ChIP-chipB_]^ji_]dWbFhecej[hh[ifedi[_dl_le• Custom Cloning and Mutagenesis• Pathway Screening• Sequence Variant Assays

9

Multiplex Histone PTM Assays

NEW: Histone PTM Multiplex Assays for Use on Luminex® Instruments
Active Motif is the ﬁrst company to provide epigenetic assays for use on the Luminex platform. We have developed
a collection of histone post-translational modiﬁcation (PTM) antibody-conjugated bead sets. These beads can be
combined together with a single lysate sample for multiplexing the interrogation of the level of histone PTMs. A
total Histone H3 antibody-conjugated bead is available for normalization of histone levels between samples.
The ﬁrst multiplex epigenetic assay
Histones are responsible for packaging DNA into nucleosomes, the basic
structural unit of chromatin. They are
subject to a variety of post-translational
modiﬁcations on the N-terminal tailsof the histone proteins, including
acetylation (Ac), methylation (Me) andphosphorylation (P), that function toregulate gene expression and thechromatin structure.

Ijh[fjWl_Z_d#f^oYe[hoj^h_d

d
ej_

I7#F;

8_

8_ej_dobWj[Z>_ijed[>)7X
H3

j_d
8_e

I7#F;

j_d
8_e

I7#F;

H3

H3

>_ijed[YedjW_d_d]FJC
Ac

P

Me3

FJC7X#Yed`k]Wj[ZX[WZ

The importance of studying histone
modiﬁcations has implications forhuman health and disease because astrong correlations have been shown toexist between speciﬁc histone modiﬁcations and human pathologies. Current
methods to study histone PTMs include
Western blot, immunohistochemistry
and genome-wide mapping. All of thesemethods are time-consuming and lackhigh throughput capabilities. Active
Motif’s Histone Modiﬁcation ELISAs
offer a solution to the issues of time andthroughput, but are limited to the analysis of a single histone PTM per ELISA.
Active Motif has partnered with Luminex®, the industry leader in multiplexing technology, to develop the ﬁrst
multiplex epigenetic assay for the studyof histone PTMs. This assay enables highthroughput processing using low sample
amounts (nanogram quantities) to interrogate multiple histone modiﬁcations
within a single well. The assay format
also offers the ability to normalize PTM
data against total histone H3 levels toevaluate relative histone PTM levels.

10

Figure 1: Schematic of the Histone PTM Multiplex Assay.

How does the assay work?
The Histone PTM Multiplex assays work
as solution-based sandwich ELISAs tointerrogate the levels of histone modiﬁcations within acid extracted cell lysatesor puriﬁed histones. Fluorescently
labeled magnetic beads, which havebeen conjugated to antibodies thattarget a speciﬁc histone PTM, are used
to bind the histone within the sample.
A biotinylated antibody speciﬁc for the
C-terminus of Histone H3 is then added
to bind the captured histone to form a“sandwich”. Streptavidin-phycoerythrin is
introduced into the reaction to bind thebiotinylated antibody and determine thesignal intensity (Figure 1).
Because each antibody-conjugatedbead set emits a unique ﬂuorescence,
antibodies for multiple histone modiﬁca-

tions can be added to the same sample
for multiplexing. Inside the Luminex
200™ or MAGPIX instruments, the identity of each bead (and it’s associatedhistone PTM) is determined based on
the emitted ﬂuorescent signal. A second
light source is then applied to the samebead to determine the magnitude of thestreptavidin-phycoerythrin signal. The
Luminex xPONENT software programprovides a real-time readout of signal as
median ﬂuorescent intensity (MFI). The
readout from the streptavidin-phycoerythrin is proportional to the amountof captured histone. When the Histone
H3 Total bead set is included in thereaction, values across samples can benormalized for histone H3 levels to show
relative amounts of each speciﬁc histone
modiﬁcation (Figure 2).

Toll Free — 1 877 222 9543

June 2013 • volume 14 • number 2)'''

Leki\Xk\[:fcZ\d`[ki\Xk\[

(,''

XZ\kp
c

Figure 3: Histone PTM Assay using all 10 bead sets in multiplex.
HeLa acid extract was tested at varying concentrations using all 10 available antibody-conjugatedbead sets in multiplex. Raw MFI values are displayed. The combination of all 10 bead sets within asingle well had no discernible effects on the assay.

('''
,''
'

\)
?*B)
.d\*

),''

>_ijed[>)DehcWb_p[Z:[j[Yj_ede\>)I'&f^

?*B)
.d

C[Z_Wd<bkeh[iY[dj?dj[di_joC<?

*'''

?*g
Xe$

#!

?*J('g_#!

\*#!

\(#!

>[BWWY_Z[njhWYj!%#YebY[c_Zjh[Wjc[dj.ª]%bWd[(«^,''e^),'e^(),e^
9cXeb

?*B0
d

'(

'&#fb[n>_ijed[FJC7iiWo

(+'''
()'''
(''''
/'''
-'''
+'''
)'''
'

?*B0
XZ

(.
()

?*Kf
kXc

C[Z_Wd<bkeh[iY[dj?dj[di_joC<?

))\*(ª]%cb *ª]%cb?*B+d(ª]%cb *ª]%cb?*B0d>)I'&f^\)>)jejWb

?*B0
d

CM
a:W

Multiplex Histone PTM Assays

),'

,''
>[BW7Y_Z;njhWYjd]

('''Figure 2: Comparison of data obtained from a Western
blot or Histone PTM Assay for amounts of H3S10ph.
To evaluate changes in total histone H3 and H3S10phlevels from untreated or colcemid-treated HeLa acidextract, a Western blot (top image) was compared witha 2-plex Histone PTM assay using Total H3 and H3S10ph
beads (bottom image). The Western blot lacks throughput capabilities, requires microgram sample quantities
to study each antibody and does not provide normalized information. The Histone PTM assay requires only
nanogram quantities of sample from which multiple
antibodies can be evaluated simultaneously in a single
well. Data can then be normalized against the total H3values to demonstrate the relative amounts of H3S10ph
in each sample.

Histone PTM Multiplex advantages• First-of-its kind – only commercially available kit to perform
multiplex histone analysis• Sensitivity – works on crude
acid extracts or puriﬁed histones
using only nanogram quantities• Multiplexing – enables analysis
of multiple histone PTM levels
within the same sample• Normalization – relative levelsof histone PTMs can be determined by normalizing against
the Histone H3 Total bead set

www.activemotif.com

What’s in the box?
The Histone PTM Multiplex Kit includes
a 96-well assay plate, a biotinylated
Histone H3 reporter antibody, streptavidin-phycoerythrin, and assay buffers.
The Histone PTM antibody-conjugatedbeads are purchased separately. This
enables the assay to be customized to ﬁt
the individual needs of each researcher.
For your convenience, a positive control
for the PTM of interest is included witheach histone PTM bead set.

All antibody-conjugated beads havebeen conﬁrmed to work when multiplexed, without any negative effects on
assay results (Figure 3). Simply use thebead sets of interest in combination
with the Histone PTM Multiplex Kit andyou will have all the reagents needed toperform the assay. For normalization ofPTM data, the Histone H3 Total-conjugated bead set is required.
To learn more about our Luminex assays,
visit www.activemotif.com/luminex.

Product

FormatCatalog No.

Histone PTM Multiplex Kit

96 rxns

33115

Histone H3 Total Ab-conjugated beads

48 rxns

33116

Histone H3K9ac Ab-conjugated beads

48 rxns

33117

Histone H3K9me1 Ab-conjugated beads

48 rxns

33118

Histone H3K9me2 Ab-conjugated beads

48 rxns

33119

Histone H3K9me3 Ab-conjugated beads

48 rxns

33120

Histone H3K4me3 Ab-conjugated beads

48 rxns

33121

Histone H3S10ph Ab-conjugated beads

48 rxns

33122

Histone H3 pan-acetyl Ab-conjugated beads

48 rxns

33123

Histone H3K27me2 Ab-conjugated beads

48 rxns

33124

Histone H3K27me3 Ab-conjugated beads

48 rxns

33125

11

Histone Peptide Array

NEW: Screen Protein Domains for Histone Binding Partners
Active Motif was the ﬁrst company to introduce an array capable of analyzing antibody and protein interactions
with histone post-translational modiﬁcations (PTMs). Our unique MODiﬁed™ Histone Peptide array offers the ability
to screen 384 different modiﬁcation combinations to evaluate cross-reactivity between individual modiﬁcations aswell as investigating the effects of adjacent modiﬁcations on binding afﬁnity. Active Motif has expanded our product offering with our new MODiﬁed™ Protein Domain Binding Kit. This assay is designed for researchers interested inscreening their protein domains (e.g. bromodomains, chromodomains, etc.) for reactivity with speciﬁc histone PTMs.

What are protein domains?
The epigenetic information that exists inthe form of post-translational modiﬁcations on histone tails is generated,interpreted and edited by proteins thatare coined “writers”, “readers” and “erasers”. There are several classes of protein
domains that inﬂuence gene regulationand chromatin remodeling by interactingwith speciﬁc histone PTMs. See Table 1
for a list of some common chromatin
remodeling protein domains.
Our current understanding of these
protein domains has been limited by thelack of appropriate tools to fully interrogate their binding speciﬁcity. Active
Motif’s MODiﬁed™ Histone Peptide Arrayprovides a solution to this problem as
it enables researchers to screen a large
panel of histone modiﬁcations in a single
experiment. The results provide informa-tion on protein domain interactions withspeciﬁc histone modiﬁcations, and also
reveals the inﬂuence that adjacent modiﬁcations may play on protein binding.
How does it work?
Active Motif’s MODiﬁed Histone
Peptide Array contains modiﬁcations
to study histone acetylation, methylation, phosphorylation or citrullination
on histones H2A, H2B, H3 and H4. Eachpeptide is spotted in duplicate on thearray to analyze the reproducibility ofthe binding events.
The MODiﬁed Protein Domain Binding
Kit was designed to be used in conjunction with the peptide arrays. The simple
assay works like a Western blot. The
array is ﬁrst incubated in blocking buffer.
Then, the His-tagged protein domain
of interest is added to the array anddetected via an anti-His tag antibody
and an HRP-conjugated secondary antibody. ECL detection is used to produce a
signal. An image of the array is captured
using a luminescent imaging system.
Free Array Analyze Software
Active Motif offers free Array Analyze
software that can be downloaded fromour website to analyze the intensity ofthe spots. Information about binding
partners can be interrogated through thesoftware program to help identify thehistone modiﬁcations associated withbinding of the protein domain (Figure 1).
We have recently updated our Array
Analyze software package to include
new features that make the softwareeasier to use and simplify the way the
results are displayed. Download a copyof our new software program today atwww.activemotif.com/modiﬁed.

DomainBromo domain

Binding Site
Acetylated lysine residues onhistones or other proteins

Function
Regulates chromatin structure and gene expression as part ofhistone acetyltransferases or chromatin remodeling factors

ExamplesTAFII250, PCAF, GCN5

Chromo domain

Methylated lysine residues on
Histone H3

Associated with the assembly of protein complexes onchromatinHP1β, MPP8, CHD1

Tudor domain

Methylated lysine or arginine
residues on Histone H3 and H4

It may be involved in RNA binding, DNA damage response andchromatin modiﬁcationJMJD2A, 53BP1, SMN

MBT domain

Methylated lysine residues on
Histone H3 and H4

The exact function is unknown, but MBT often appears as repeats inproteins associated with transcriptional repression

L(3)MBTL, CGI-72

Table 1: Examples of some common protein domain classes and their associated binding speciﬁcity and function.

12

Toll Free — 1 877 222 9543

June 2013 • volume 14 • number 2

Histone Peptide Array
Figure 1: Analysis of the JMJD2A protein
domain using the MODiﬁed Protein Domain
Binding Kit and the Modiﬁed Histone Array.
The His-tagged Tudor domain protein,
JMJD2A, was used at a 50 nM concentration
with the MODified Protein Domain Binding
Kit and the MODified Histone Peptide Array.
An image of the array was captured using achemiluminescent imaging system.
Top: JMJD2A array image in which the grid
overlay on the left half demonstrates thealignment performed by the Array Analyze
software program for spot identification.

8_dZ_d]If[Y_\_Y_joe\@C@:(77l[hW][?dj[di_jo

(
'%/
'%-

8[[`k`fef]df[`]`ZXk`fejXkI)_Xm\c`kkc\\]]\ZkfeY`e[`e^XkB).d\*J`e^c\B).d\*df[`]`ZXk`fe'%+

8[[`k`fef]J)/g_`e_`Y`kjY`e[`e^

'%)

?+

le
?+ df
B) [
'd
?+ \*
B(
-X
?
?
+
?+ B +B Z
B( (-X)XZ ZB ()XZ
B( )'d-X
ZB \*)'?* d\*
le?* df[
B)?* .d\I) *?* -d\I) )j-d?*I) ?*I \)X?* -d\ )-:I) )j `ki-d B)?* \)X .d\I) B) *
-: .d
?*
`
I) ?* kiB) \*
B?* d\) ).d .d\*I) jB \* -d ). J)/\) d\ g_XB *J).d )/
\* g_
J)
/g
_

'

CE:_\_[Z7hhWoF[fj_Z[?Z[dj_jo

MODiﬁed Array Labeling Kit
In addition to the new Protein Domain
Binding Kit, the MODiﬁed Histone
Peptide Arrays can also be used toscreen antibodies for cross-reactivity.
The MODiﬁed™ Array Labeling Kit
provides all the reagents needed for theblocking, washing and chemiluminescentdetection of the array. HRP-conjugatedsecondary antibodies for use with either
rabbit or mouse primary antibodies, anda positive control antibody that recognizes the c-Myc control spot printed
on the peptide array are also included.
The c-Myc control antibody can be
combined with the antibody you arescreening to assist with spot identiﬁcation within the Array Analyze software.

www.activemotif.com

Bottom: Graphical analysis of selected datafrom the Array Analyze software program which
was used to calculate the spot intensity ofbinding events for the JMJD2A protein domain.
The average intensity was calculated for each
duplicate on the left and right side of the arrayand graphed for the peptides listed.
The data shows the specificity of the JMJD2A
domain for Histone H4K20me3 and H3K27me3(green arrow). The presence of additional modifications within the same peptide had varying
influences on spot intensity. There was relatively little change when the H3K27me3 modification was present together with modificationsat R26 (copper arrows). But, when the S28ph
modification was adjacent to K27me3, bindingof the JMJD2A protein domain was inhibited
greatly (red arrows).

Active Motif offers a broad selection of recombinant proteins,
including Bromodomains, HATs, HDACs, HMTs and HDMs, which can beused to study functional changes and binding speciﬁcity on theMODiﬁed Histone Peptide Array. To see a full list of available proteins,please visit us at www.activemotif.com/proteins.

Product

FormatCatalog No.

MODiﬁed™ Histone Peptide Array

1 array
5 arrays

13001
13005

MODiﬁed™ Protein Domain Binding Kit

5 rxns13007

MODiﬁed™ Array Labeling Kit

5 rxns13006

13

DNA Methylation

MethylCollector™ Ultra for the Most Sensitive Enrichment of Methylated DNA
Active Motif’s popular MethylCollector™ Ultra Kit is the fastest, most sensitive and speciﬁc kit available for enrichment of CpG methylated dinucleotides from fragmented genomic DNA. The patented MIRA (Methylated CpG Island
Recovery Assay) technology combined with a magnetic bead-based protocol enables the most efﬁcient methylated
DNA enrichment method possible, with results in less than half the time required for MeDIP.
MethylCollector Ultra for the fastest
enrichment of methylated DNA
Active Motif’s MethylCollector Ultra Kit
provides the most rapid and sensitive
method for enrichment of methylatedDNA. The kit’s design is based on thepatented MIRA technology that uses a
puriﬁed MBD2b/MBD3L1 protein complex to bind methylated DNA. This highafﬁnity binding method provides greater
enrichment than other MBD capture or
antibody immunoprecipitation (MeDIP)
methods (Table 1). MethylCollector Ultrarequires as little as 1 ng (~200 cells) ofinput DNA and enables detection of
methylation from DNA fragments
containing as few as 5 methylated CpGs.
Magnetic capture allows more rapid andefﬁcient recovery of methylated DNA.
Recovered dsDNA is compatible withvarious downstream applications including Next-Gen Sequencing (Figure 1).

MethylCollector™ Ultra (Cat. No. 55005)

MeDIP Kit (Cat. No. 55009)

Input material: 1 ng - 1 µg

Input material: 100 ng - 1 µg–

Denature DNA for 10 minutes at 95°C

Combine DNA with MBD2b/MBD3L1 protein mix
and nickel-coated magnetic beads for 1 hour at 4°C

Combine DNA, antibody, bridging antibody andmagnetic beads, incubate overnight at 4°C

Wash beads 4 times using the included magnet

Wash beads 5 times

Elute methylated DNA at 50°C for 30 minutes

Elute methylated DNA at 4°C for 15 minutes

Add Proteinase K Stop Solution

Add Neutralization Buffer

DNA clean up by phenol/chloroform and ethanol
precipitation, or with puriﬁcation columns

DNA clean up by phenol/chloroform and ethanol
precipitation, or with puriﬁcation columns

DNA is ready for analysis

DNA is ready for analysis

Total time required: < 3 hours

Total time required: > 7 hours

Table 1: Comparison of the MethylCollector™ Ultra process to MeDIP.

NEW: MethylCollector Ultra-Seq Services Now Available
Active Motif has now expanded its
offerings to include end-to-end MethylCollector™ Ultra-Seq Epigenetic Services.

Simply submit your cells or DNA andreceive fully analyzed genome-wide
methylation data in just 6-8 weeks.

MIRA-Seq Service includes:• DNA isolation from cells or tissues• Methylated DNA enrichment using
MethylCollector Ultra• qPCR analysis of positive andnegative control sites• Next-Gen Library generation• Sequencing of at least 30 million
tags using the Illumina HiSeq
platform• Analysis: mapping, peak calling,visualization ﬁles and Excel output

C[j^ob9ebb[YjehKbjhW

14

For complete details on this and otherservices offered, please visit us atwww.activemotif.com/services.

9ecf[j_jehC

THRAP3

Clorf113
p34.3

36,760,000

36,780,000

36,800,000

36,820,000

STK40

36,840,000

LSM10

Figure 1: MethylCollector Ultra is more sensitive than other MBD-based enrichment methods.
The performance of MethylCollector Ultra was compared to that of an alternative MBD capture kit. Methylated DNA was
isolated from 1 µg of human PBMC DNA using both enrichment kits. The enriched DNA was sequenced using the Illumina
HiSeq platform. Regions of enrichment from a 1.2 Mb region of the genome are shown. The MethylCollector Ultra technology
detected more methylated regions and the peaks were of greater intensity than those detected by the competitor’s kit.

Toll Free — 1 877 222 9543

June 2013 • volume 14 • number 2

Stem Cells

NEW: CDy1 Fluorescent Dye for Identiﬁcation and Imaging of Live Stem Cells
Active Motif’s new Stem Cell CDy1 Dye was developed in a collaboration with scientists at the Agency for Science,
Technology and Research (A*STAR) in Singapore as a live cell imaging probe that is selective for stem cells. CDy1 offersa faster, higher throughput, more cost-effective way to identify and isolate stem cells compared to conventional
techniques, such as alkaline phosphatase or immunostaining methods. Furthermore, unlike techniques that requirecell manipulation or ﬁxation, CDy1 does not impact cell growth, morphology or differentiation, making it ideal for
FACS sorting and isolation of puriﬁed stem cell populations. Because the integrity of cells is preserved, additional
experiments and immunostaining can be performed following CDy1 staining.

Why use the Stem Cell CDy1 dye?
Aside from ease of use and higher
throughput, CDy1 offers other advantages over conventional methods for stem
cell identiﬁcation. CDy1 staining does
not affect the differentiation potentialof stem cells. As this staining method
does not require the sacriﬁce of cells,
additional experiments or immunostaining can be performed following CDy1staining. Another advantage of this novel
staining method is that it eliminates theneed to generate a reporter system toscreen for stem cell marker expression.
For reprogrammed cells, CDy1 is known
to identify iPS cells earlier than genetic
reporter systems (Kang et al. (2011)
Nature Protocols 6: 1044-1052).

www.activemotif.com

How are researchers using CDy1?
Recent publications highlight the advantages of using CDy1 for identiﬁcation,enrichment and characterization of various stem cell types including embryonic,
reprogrammed, neural and cancer stem
cells. For an up-to-date list of publications, information and data on the CDy1
Dye, visit www.activemotif.com/cdy1.

'A
.&&
,&&
*&&
(&&
&

Recent CDy1 Publications:• Hawley et al. (2013) Am J Hematol. 88: 265-272.
• Vukovic et al. (2013) Stem Cells Dev. Apr 27.
• Vendrell et al. (2012) Stem Cell Res. 9: 185-191.
• Kang et al. (2011) Nature Protocols 6: 1044-1052.
• Im et al. (2010) Angew. Chem. Int. Ed. Engl. 49:
7497-7500.

F^Wi[

9:o'

c;I9
C;<

II9

Novel stem cell dye for live cells
Active Motif’s Stem Cell CDy1 Dye is aneasy-to-use live cell dye with a fast andsimple cell staining protocol for stem
cell identiﬁcation and isolation fromfeeder cells or mixed cultures. The Stem
Cell Dye is simply added to the culture
media. Following a 1 hour incubation,
the cells are washed and prepared for
imaging by ﬂuorescence microscopy or
FACS analysis. The spectral properties of
the CDy1 dye enable the detection ofstained cells using common ﬂuorescentﬁlter sets. For microscopy, TRITC/Cy3
ﬁlter sets can be used, while a 488 nm
laser and a PE-Texas Red ﬁlter are used
for ﬂow cytometry.

'&&

'&'

'&(

F;#J[nWiH[Z

'&)

'&*

Figure 1: FACs data of CDy1 staining on mESCs and MEFs.
Flow cytometry dot-plot image overlay of pure populations of Stem Cell CDy1 stained mouse ESCs (mESC) in
red and mouse embryonic fibroblasts (MEF) in blue.
The cells were analyzed using side scatter (SSC) and a
PE-Texas Red channel. CDy1 enables clear identificationand isolation of embryonic stem cells.

EYj*

Figure 2: CDy1 Staining Coincides with Expression of Stem Cell Markers.
Mouse embryonic stem cells (mESCs) grown on a mouse embryonic fibroblast (MEF) feeder layer were stained without
fixation with Stem Cell CDy1 dye. CDy1 staining was visualized by fluorescent microscopy. The cells were then fixed in
4% paraformaldehyde, stained with Oct4 antibody and visualized with FITC-conjugated secondary antibody. The images
show that mESC aggregates, but not MEF feeder cells, stain positive for both CDy1 (red) and Oct4 (green). Scale bar, 100 µm.
The images were kindly provided courtesy of Dr. Y-T Chang at the National University of Singapore, Republic of Singapore.

Product

FormatCatalog No.

Stem Cell CDy1 Dye

50 µl

14001

15