THE NEWSLETTER OF ACTIVE MOTIF — August 2008 • volume 9 • number 2

2 NEW: Recombinant Histones with Site- & Degree-specific Methylated Lysines
3 Create Chromatin In Vitro to Reveal Regulatory Mechanisms
4 NEW: High-throughput ChIP with ChIP-IT™ Express HT

IN
THIS
ISSUE

5 ChIP Accessory Kits and Reagents
6 Double Destabilized Luciferase Provides Better Response & Sensitivity
7 Easily Compare CpG Methylation in Various DNA Samples
7 Reproducible Bisulfite Conversion for Accurate Analysis of Methylated DNA
8 NEW: Assays for IRF-3 & -7 and SREBP1 Activity
9 Lower Background Co-IP of Both Nuclear & Whole-cell Complexes
10 Rapid, Sensitive Assays for HAT & HDAC Activity and Inhibitor Compounds
11 NEW: Antibodies to Study Transcription, Histones and Chromatin Biology

Methylated Histones

NEW: Recombinant Histones with Site- & Degree-specific Methylated Lysines
Active Motif is pleased to announce the ﬁrst release in its new collection of Recombinant Histones, which focuses on
Histone H3 proteins with site-speciﬁc mono-, di- and tri-methylated lysines. These proteins enable you to investigate
how speciﬁc lysine methylations inﬂuence nucleosome remodeling and the binding of associated chromatin proteins.
Histones & chromatin structure
Methyl-lysine residues in nucleosomal
histones are thought to mediate
interactions with the protein complexes
involved in regulating transcription,
replication and DNA repair. In order toinvestigate these complex functionalquestions, histones with speciﬁc methylation states are required to evaluate
which methylation patterns are key toregulatory processes. Active Motif ispleased to be the ﬁrst to offer methylated histone H3 proteins that can beused in nucleosome remodeling assays
to investigate the implication of speciﬁcmethylation on chromatin function.

How is the methylation state made?
Recombinant methylated histones
are created via a chemical alkylation
reaction* that introduces an analog ofmethyl lysine. This speciﬁc chemicaltreatment enables the site and degree ofmethylation to be controlled precisely,
so each methylation reaction is over 99%
complete, as veriﬁed by high-resolution
ESI-TOF mass spectrometry. All recombinant histones are also conﬁrmed by dot
blot or immunoblot (Figure 2). As the

methylation state closely mimics natural
methylation, these recombinant histones
are perfect for any functional assay.
Order today!
Visit our website for more information on how to use these recombinanthistones as a standard for your assays or
as a building block for speciﬁc in vitro
chromatin assays (Figure 1).
*Patent pending.

Methylated Histone H3
Active Motif currently offers recombinant histone H3 proteins mono-, di- andtri-methylated at lysines 4, 9 & 27. Visit
our website to stay current with thelatest releases of methylated histones.

'

(

)

*

+

,

-

.

/

'&

''

'(

Figure 2: Western blot analysis of mono-, di- and tri-methylated Recombinant Histone H3.
One µg Recombinant Histone H3 (lanes 1, 5 & 9), 1 µg Recombinant Histone H3 monomethyl Lys9 (Lanes 2, 6 & 10),
1 µg Recombinant Histone H3 dimethyl Lys9 (Lanes 3, 7 & 11) and 1 µg Recombinant Histone H3 trimethyl Lys9 probed with
anti-Histone H3 monomethyl Lys9 (Cat. No. 39249) (Lanes 1-4), anti-Histone H3 dimethyl Lys9 (Cat. No. 39239) (Lanes 5-8)
and anti-Histone H3 trimethyl Lys9 (Cat. No. 39161) (Lanes 9-12).

1

2

3

Figure 1: Ordered spacing of nucleosomes after
enzymatic digestion of assembled chromatin.
Recombinant Histone H3 dimethyl Lys9 (4.5 µg) wasused to generate chromatin in vitro using the Chromatin
Assembly Kit (Cat. No. 53500). One µg of assembled
chromatin was digested for 2 minutes (Lane 2) and4 minutes (Lane 3). Lane 1 is 100 bp marker.

2

Product

FormatCatalog No.

Recombinant Histone H3 (C110A)

50 µg

31207

Recombinant Histone H3 monomethyl Lys4

50 µg

31208

Recombinant Histone H3 dimethyl Lys4

50 µg

31209

Recombinant Histone H3 trimethyl Lys4

50 µg

31210

Recombinant Histone H3 monomethyl Lys9

50 µg

31211

Recombinant Histone H3 dimethyl Lys9

50 µg

31212

Recombinant Histone H3 trimethyl Lys9

50 µg

31213

Recombinant Histone H3 monomethyl Lys27

50 µg

31214

Recombinant Histone H3 dimethyl Lys27

50 µg

31215

Recombinant Histone H3 trimethyl Lys27

50 µg

31216

Toll Free — 1 877 222 9543

Chromatin AssemblyAugust 2008 • volume 9 • number 2

Create Chromatin In Vitro to Reveal Regulatory Mechanisms
Active Motif’s Chromatin Assembly Kit enables you to generate chromatin
in vitro from your linear or supercoiled DNA. It yields chromatin that closely
mimics natural in vivo chromatin, so you can discover which histone modiﬁcations and associated proteins are crucial to regulation of your target.
A simple way to generate chromatin
Now you can investigate regulation ofyour gene of interest in its native form
by assembling it into chromatin using
Active Motif’s Chromatin Assembly Kit.
The kit includes all the recombinant
proteins, core histones, buffers andATP-utilizing factors needed to generate
chromatin in vitro from your DNA, andalso to verify successful assembly. Highquality chromatin with more than six
regularly spaced nucleosomes is made byadding the supplied components to 1 µgof your linear or supercoiled DNA, thenincubating for 4 hours. A simple partial
enzymatic digestion of the resulting
chromatin conﬁrms the ordered spacingof nucleosomes (Figure 1).
M

1

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3

assembled state often cannot reveal themechanism of transcriptional activation
or repression with the associated factors
and relevant histone modiﬁcations.
However, properly assembled chromatinwith regularly ordered nucleosomes is an
excellent substrate for subsequent assays such as in vitro transcription assays,
histone acetyltransferase (HAT) assays(Figure 2) and ChIP (Figure 3).

- +

p53

p300
p53

4
H3
H2B
H2A
H4

1

2

HAT Assay
Figure 2: HAT assay using assembled chromatin.
Chromatin was in vitro assembled with p300 and acetylCoA, in the presence (+) or absence (-) of p53 at 30°C for
30 minutes. The sample was then analyzed on an
18% SDS-PAGE gel and visualized following fluorography.
Figure 1: Enzymatic digestion of assembled chromatin.
Chromatin assembled from 1 µg samples of circular DNA(Lanes 1 & 2) and linear DNA (Lanes 3 & 4) were digested
for 2 and 4 minutes, respectively, deproteinated, phenol/
chloroform extracted and run on an agarose gel. Eachsample type resulted in regularly spaced nucleosomes.

Why bother to make chromatin?
When DNA sequences are assembled
into chromatin by ATP-dependent
factors, the resulting structure closely
resembles the natural chromatin conﬁguration. DNA that is in either a bare or un-

www.activemotif.com

More natural for true results
Now you can use recombinant degreeand site-speciﬁc methylated histones(Page 2) to create chromatin that closely
mimics the in vivo form, so you can conﬁrm results and be certain about which
factors are critically involved in regulation of your target sequence.

Chromatin Assembly Kit advantages• Generate chromatin from linear orsupercoiled DNA• ATP-dependent method results
in an extended array of regularly
spaced nucleosomes• Easy protocol – simply incubate thekit components with your DNA• Produces an excellent substrate forvarious gene regulation experiments

Input
AP-1

anti-hexahistidine- + - +

Figure 3: ChIP of in vitro assembled chromatin.
In vitro ChIP was performed using in vitro-assembled
chromatin containing an AP-1 binding site in thepresence of p300 and acetyl-CoA, with or withouthexa-histidine-tagged AP-1. The ChIP was performed
with a hexahistidine antibody and PCR was conductedwith primers flanking the AP-1 binding site.

Complete kit ensures your success
The kit includes recombinant h-NAP-1
chaperone protein, ACF assembly complex, HeLa core histones, and the buffers
and enzymes necessary to generateassembled chromatin from your input
DNA. Control Supercoiled DNA is also
provided. To verify that your chromatin
assembly reaction has been successful,reagents are provided to perform aneasy partial enzymatic digestion to visualize the regular spacing of nucleosomesby agarose gel electrophoresis (Figure 1).
Start assembling chromatin now!
The Chromatin Assembly Kit makes it
easy for you to assemble chromatin on
your sequence of interest to investigate
gene regulation in a context that closely
resembles in vivo chromatin. For more
information, please give us a call.

Product

FormatCatalog No.

Chromatin Assembly Kit

10 rxns

53500

3

High-throughput ChIP

NEW: High-throughput ChIP with ChIP-IT™ Express HT
Active Motif now makes it easy for you to perform your ChIP experiments on a high-throughput scale with theintroduction of ChIP-IT™ Express HT. Based on the innovative ChIP-IT Express Kit, it uses the time-saving magnetic beadmethod for faster and cleaner ChIP experiments and enables the processing of up to 96 ChIP reactions at once.
ChIP-IT Express HT Advantages• Process up to 96 ChIP reactions• Faster plate-based protocol• Fewer cells required per ChIP
• Compatible with ChIP-chip and
ChIP-seq methodologies
High-throughput ChIP
If you need to perform many ChIP
experiments at once, the new ChIP-IT
Express HT kit is your solution. It combines the time-saving, magnetic beadbased protocol of ChIP-IT Express witha high-throughput, 96-well microplatebased format. With ChIP-IT Express HT
you can rapidly and efﬁciently process
up to 96 ChIP reactions at a time. ChIP-IT
Express HT is compatible with our
enzymatic and sonication-based shearing
kits for chromatin preparation, as well aswith the ChIP-IT Control Kits.
Improved ChIP
Chromatin Immunoprecipitation (ChIP)
is an important technique, enabling theidentiﬁcation of the in vivo sites of chromatin binding proteins, or localization ofhistone modiﬁcations to speciﬁc regionsof the genome. Recent improvements in
ChIP protocols and reagents have madeChIP experiments faster and easier toperform, as well as more successful. In
addition, the lower background enabledby improved ChIP techniques has madeit possible to perform ChIP with fewer
cells, and to perform more ChIP reactions at one time. Active Motif has beenat the forefront of these advances with
the introduction of magnetic beads for
ChIP in its ChIP-IT Express Kit.

4

Figure 1: Chromatin IP performed on HeLa chromatinusing ChIP-IT Express HT.
PCR carried out using primers specific for the GAPDH
gene. Lanes 1-4, ChIP using 2 µg anti-RNA Pol II antibody.
Lanes 5-8, ChIP using normal mouse IgG as a negative
control. Lane 9, no DNA control. Lane 10, input DNA
control.

FREE 96-well magnetic sorter!
Order a ChIP-IT Express HT and receivea free 96-well magnetic sorter. Available
while stocks last.

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

Positive controls ensure success
Because interpreting ChIP data can be
difﬁcult, Active Motif has developed acomplete line of control kits to help you
troubleshoot your assays. To provide
you with controls that are appropriate
for your experimental model, we offer
ChIP-IT control kits for human, mouseand rat.
Take ChIP to the next level
For additional information on the new
ChIP-IT Express HT kit go towww.activemotif.com/htchip. Also,
please browse our complete line of
ChIP-validated antibodies atwww.activemotif.com/chipabs.

Product

FormatCatalog No.

ChIP-IT™ Express HT

96 rxns

53018

ChIP-IT™ Express

25 rxns

53008

ChIP-IT™ Express Enzymatic

25 rxns

53009

ChIP-IT™ Protein G Magnetic Beads

25 rxns

53014

Toll Free — 1 877 222 9543

August 2008 • volume 9 • number 2

Chromatin Immunoprecipitation

ChIP Accessory Kits and Reagents
Active Motif offers a broad range of reagents and accessory kits tocomplement the ChIP-IT™ line of ChIP kits. These products will help you
troubleshoot your ChIP experiments and make them more reproducible.
Superior ChIP-IT magnetic beads
Whether you are analyzing your ChIP
results by PCR, ChIP-chip or ChIP-seq,
having low non-speciﬁc background isalways desirable. In the past, researchers have successfully used protein G
agarose beads in their ChIP experiments.
However, it is widely known that the
agarose matrix itself non-speciﬁcallyenriches for proteins due to the intrinsic electrostatic properties of agarose.
The result is reduced assay sensitivity,
increased data interpretation times orfailed experiments. In an effort to avoid
these problems, researchers have historically had to pre-block the agarose beads
and increase the number of wash steps
in their ChIP protocol. When you use Active Motif’s ChIP-IT Protein G Magnetic
Beads there’s no need to waste yourtime and money performing extra steps.
This is because the protein G is directly
conjugated to a magnetic bead that will
not bind non-speciﬁcally to proteins inyour chromatin mix. Additionally, ChIP-IT
Protein G Magnetic Beads are ready touse and will save you signiﬁcant amounts
of process time. If you’re not looking
for a complete solution like our ChIP-IT
Express Kits, why not incorporate ourChIP-proven magnetic beads into yourin-house ChIP method to improve results
and save time?
ChIP-IT Control Kits
ChIP is an enrichment of DNA boundby a particular protein, not a complete
puriﬁcation of the protein-boundDNA. As a result, ChIPs are unavoidably
contaminated with chromatin boundwww.activemotif.com

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non-speciﬁcally. During PCR analysis,
this contaminating DNA can lead tofalse positive PCR products, making datainterpretation difﬁcult. Active Motif offers a variety of control options to help
eliminate false positives. You can orderthe original ChIP-IT Kits with or without
human controls, or you can choose toorder ChIP-IT Express and add on thecontrol kit for your species of interest.
The ChIP-IT Control Kits provide positive
and negative control antibodies andpositive control PCR primers, PCR buffer
and a convenient DNA loading dye thatmakes your PCR reactions ready forloading on an agarose gel straight fromthe thermocycler (Figure 1).

ing Cocktail to quickly and easily shear
chromatin. As enzymatic shearing issolely time and temperature dependent,
the inconsistencies associated withsonication are eliminated, which helps toimprove your ChIP results.

Simplified chromatin shearing
For ChIP experiments to be successful,chromatin must ﬁrst be sheared to 2001000 bp fragments. Traditionally, shearing
has been performed by sonication,
which is effective, but it can be time
consuming and difﬁcult to optimize. To
eliminate the problems associated withsonication, Active Motif has developed
a more robust and user-friendly method
to shear chromatin for ChIP. This method
uses our proprietary Enzymatic Shear-

Ready-to-ChIP Chromatin
For your convenience, Active Motif
offers Ready-to-ChIP Chromatin. Readyto-ChIP Chromatin has been optimallysheared by sonication and validated in
ChIP. As a result, you can more easily
validate your own antibodies and primer
sets. It can be used in conjunction withall of the ChIP-IT Kits and controls, so
you can be certain the only variable
in testing an antibody for ChIP is theantibody itself.

Figure 1: ChIP with the ChIP-IT™ Control Kit - Mouse
Chromatin IP using ChIP-IT Express (Cat. No. 53008)
performed on mouse macrophage 4/4 cells, amplifying
a region of the EF1α gene promoter. Lane 1: molecularweight ladder. Lanes 2 and 4: RNA pol II mAb (Cat. No.
39097). Lanes 3 and 5: negative IgG control. Lane 6: noprimary antibody. Lane 7: input chromatin control. Lane
8: no DNA control.

Product

FormatCatalog No.

ChIP-IT™ Protein G Magnetic Beads

25 rxns

53014

ChIP-IT™ Control Kit – Human

5 rxns

53010

ChIP-IT™ Control Kit – Mouse

5 rxns

53011

ChIP-IT™ Control Kit – Rat

5 rxns53012

Ready-to-ChIP HeLa Chromatin

10 rxns

53015

ChIP-IT™ Shearing Kit

10 rxns

53002

Enzymatic Shearing Kit

10 rxns

53005

5

Luciferase Reporter Gene Assay

Double Destabilized Luciferase Provides Better Response & Sensitivity
16

pRR-HighpRR-Low
pGL3
12Fold induction

RapidReporter®* is the only luciferase reporter gene assay that utilizes bothmRNA and protein destabilizing elements. This greatly reduces background,increasing both the magnitude of the response that can be measured after
stimulation or repression, and the speed in which the assay can measurechanges in transcription (both increases and decreases).

8

4Standard luciferase assays are limited by
the fact that basal activity of the cloned
promoter results in accumulation ofboth the luciferase mRNA and protein.
The slow clearance rate of these preexisting molecules substantially delays
and dilutes the measurable response.
Thus, a large proportion of the luciferase
measured is derived from transcriptionthat took place before the test agent waseven added. As a result, in standard assays transient or relatively minor effects
are hidden and kinetics are inaccurate.
Consequently, drugs or treatments thatare disregarded because they have little
or no detectable effect may often elicit
a 10-fold or more change in transcription.1

additional 33% compared to vectors with
only protein-destabilizing elements.2

A good start, but not the end
In trying to address the problem of long
half-life proteins, some vectors include
protein-destabilizing elements, whichcause the luciferase to degrade more
rapidly. Destabilizing the protein, however, only partly addresses the problem,
as clearance rates are also dependent on
the half-life of the mRNA. As long as thereporter mRNA is intact, it continues to
produce new reporter protein.

Product

FormatCatalog No.

RapidReporter® Gaussia Luciferase Assay

100 rxns
1000 rxns

33001
33002

RapidReporter® pRR-High vector
RapidReporter® pRR-High Assay

10 µg
100 rxns

33003
33004

RapidReporter® pRR-Low vector
RapidReporter® pRR-Low Assay

10 µg
100 rxns

33005
33006

RapidReporter® pRR-High-CRE vector
RapidReporter® pRR-High-CRE Assay

10 µg
100 rxns

33007
33008

RapidReporter® pRR-High-GR vector
RapidReporter® pRR-High-GR Assay

10 µg
100 rxns

33011
33012

RapidReporter® pRR-High-IRF-1 vector
RapidReporter® pRR-High-IRF-1 Assay

10 µg
100 rxns

33017
33018

RapidReporter® pRR-High-NFκB vector
RapidReporter® pRR-High-NFκB Assay

10 µg
100 rxns

33009
33010

RapidReporter® pRR-High-STAT1 vector
RapidReporter® pRR-High-STAT1 Assay

10 µg
100 rxns

33015
33016

RapidReporter® pRR-High-STAT3 vector
RapidReporter® pRR-High-STAT3 Assay

10 µg
100 rxns

33013
33014

Eliminate both causes of backgroundTo solve the problem, RapidReporter
utilizes vectors that include both protein
AND mRNA destabilizing elements. Suchdouble destabilizing vectors have beenshown to reduce luciferase half-life an
REFERENCES

1. Voon, C. et al. (2005) Nucleic Acids Research 33(3): e27.
2. Almond, A. et al. (2004) Promega Notes 87: 18-22.

600Measure what’s actually happening
The use of double destabilizing vectors
makes RapidReporter more sensitive andresponsive, so you can detect smallerchanges and get more accurate real-timemeasurement than with non-destabilized reporter gene assays, or those thatdestabilize the protein only (Figure 1).
Pre-made vectors & complete kits
In addition to empty RapidReporter
vectors in two different stringencies,
Active Motif offers vectors that containwidely studied promoters. All are avail-

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Hours after addition of isoproterenol

Figure 1: More accurate kinetics, higher fold induction.
293 cells transiently transfected with pRR-High-CRE,
pRR-Low-CRE and a pGL3 vector containing CRE wereplated onto 96-well plates. Twenty-four hours posttransfection, cells were stimulated with 4 µM isoproterenol, then measured for Gaussia (pRR vectors) and firefly
luciferase activities (pGL3 vector).

able separately or in complete assay kits,
which also include a positive control
vector and buffers & substrate optimized for Gaussia luciferase. To learn
more about the only reporter assay withdouble destabilization, please give us acall or visit www.activemotif.com.

* RapidReporter is covered under U.S. Patent No. 7,157,272 and various other patents worldwide and is sold underlicense granted by GeneStream Pty Ltd. Purchasers are subject to a Limited-use License; please contact Active
Motif’s Technical Services for details, or download a copy from our web site. RapidReporter is a registeredtrademark of GeneStream Pty Ltd.

Toll Free — 1 877 222 9543

DNA Methylation

August 2008 • volume 9 • number 2

Easily Compare CpG Methylation in Various DNA Samples
MethylCollector™ provides a fast, efﬁcient protocol for isolating andcomparing CpG-methylated DNA in cell or tissue samples. The kit uses a
recombinant Methyl-binding protein (MBD2b) to capture DNA, rather thanantibody-based immunoprecipitations, greatly improving assay sensitivity.
MethylCollector advantages• Rapid – simple protocol iscompleted in less than 4 hours• Adaptable – enables detection ofsamples from 5 ng to 1 µg of DNA• Verified – positive control DNA andPCR primers ensure success
The MethylCollector method
In MethylCollector, His-tagged MBD2b
speciﬁcally binds to CpG-methylated
DNA fragments prepared by sonicationor enzymatic digestion. These proteinDNA complexes are captured withnickel-coated magnetic beads andwashed with a stringent high-salt buffer
to remove DNA fragments with little orno methylation. Ready-to-use methylated DNA is then eluted (Figure 1). Because
MethylCollector is highly efﬁcient, you
can analyze the methylation state of any
speciﬁc locus on genomic DNA isolated
from less than 800 cells (~5 ng DNA).
MethylCollector applications
MethylCollector enables many powerfulapplications, including rapid screening of
the methylation status of multiple lociin tumor tissue or cells and detectingchanges in DNA methylation in other

0

1

MBD2b (µg)
0
1
1

2

3

4

Input

5

6

7

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Figure 1: Isolation of Fully Methylated Jurkat DNA.
MethylCollector was used to isolate methylated DNA
from 100 ng genomic DNA (lanes 2 & 4) and Fully
Methylated Jurkat DNA (lanes 3 & 5), with (lanes 4 & 5)
or without (lanes 2 & 3) inclusion of 1 µg of the kit’s
His-tagged MBD2b protein. These samples and input
genomic DNA (lane 6) and input Fully Methylated Jurkat
DNA (lane 7) were then PCR amplified with the BRCA1
primers. A positive result is observed only with Fully
Methylated Jurkat DNA captured by the kit’s His-tagged
MBD2b (lane 5). Lane 8 is a water-only PCR control.

situations, such as normal cellular differentiation and aging. To ﬁnd out more,
please visit www.activemotif.com.

Product

FormatCatalog No.

MethylCollector™

25 rxns

55002

Fully Methylated Jurkat DNA

10 µg

55003

Reproducible Bisulfite Conversion for Accurate Analysis of Methylated DNA
The MethylDetector™ Bisulﬁte Modiﬁcation Kit makes DNA methylation
analysis fast and efﬁcient by providing optimized reagents, time-saving DNA
puriﬁcation columns and positive control PCR primers for assay validation.
Proven controls verify your success
DNA methylation analysis often uses
bisulﬁte to convert unmethylated
cytosines to uracils, leaving methylated
cytosines unchanged. The DNA is thenPCR ampliﬁed and analyzed by sequencing or restriction digest, which canbe costly and time-consuming. Thus,
conﬁrming that conversion was successful before analyzing the samples is a big
beneﬁt. To that end, the MethylDetector
Kit provides positive control PCR primers
speciﬁc for bisulﬁte-converted DNA, so
you can conﬁrm the conversion worked
before starting the analysis (Figure 1).

www.activemotif.com

Advantages• Reproducible – 99% conversion
efﬁciency• Flexible – use high G/C content
sequences and uncut DNA• Easy – DNA puriﬁcation columns
mean no more precipitations
Try MethylDetector today
MethylDetector will speed and simplify
your methylation analysis. Learn more atwww.activemotif.com.

M

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4

5

500 bp400 bp300 bp200 bp100 bpFigure 1: Reproducible conversion by MethylDetector.
MethylDetector was used for bisulfite conversion of
3 different DNA samples (Lanes: 1-3) and a control with
no DNA (Lane: 4). PCR was performed on these samples
and an unconverted DNA control (Lane: 5) using the kit’s
control PCR primers. The presence of PCR product inonly the converted samples demonstrates the efficiency
and reproducibility of the MethylDetector Kit.

Product

FormatCatalog No.

MethylDetector™

50 rxns

55001

7

TransAM Transcription Factor Assays

NEW: Assays for IRF-3 & -7 and SREBP1 Activity
TransAM™ Kits are fast and simple DNA-binding ELISAs in a convenient format with high sensitivity and speciﬁcity forover 40 targets. Now TransAM, the most published alternative to EMSA, is available for IRF-3 & IRF-7 and SREBP1.
The TransAM method
TransAM Kits use a unique plate-basedformat to capture activated transcription factors, which are analyzed using anantibody speciﬁc to your isoform of interest. TransAM Kits are non-radioactive
and offer up to 100-fold more sensitivity
than traditional gelshift techniques, soeven the smallest changes in transcription factor levels can be detected.
How it works
TransAM Kits assay for transcription
factor activity by capturing the activatedfactor with a double-stranded oligonucleotide containing the target-speciﬁc
consensus sequence that is bound to
a 96 strip-well plate. When nuclear
extracts from tissue or cell samples
are added to the plate, the activatedtranscription factor binds the consensus
sequence on the plate. Next, a primary
antibody speciﬁc to the transcription
factor is added which is subsequently
detected by an HRP-conjugated secondary antibody and developed to give acolorimetric readout.
TransAM IRF-3 & 7
Studying Interferon Regulatory Factors(IRFs) and their involvement in host defense through regulation of immune responses, cell growth and hematopoietic
development can be difﬁcult. Accuratemonitoring of IRF activation is now
easier with TransAM Kits. Kits are available for mouse and human IRF-3 andhuman IRF-7.

8

Figure 1: TransAM Kit citations.
Using HighWire Press, http://highwire.stanford.edu,
a comparison was made by searching for citations ofTransAM from Active Motif versus the tradenames of allcompetitor kits combined. TransAM is clearly the leader.

Figure 2: TransAM SREBP1 activation.
0.6 to 10 µg of Cos-7 (SREBP1 transfected) nuclear extract(black bars) and mock-transfected Cos-7 nuclear extracts(gray bars) were assayed per well. .

TransAM citations
TransAM is the most published ELISA
assay for transcription factor activation(Figure 1). The combination of a fast,
user-friendly format with high sensitivity and speciﬁcity makes it easy to see
why over 800 citations are available forTransAM Kits.

protein is cleaved to release the N-terminal portion to enter the nucleus andactivate transcription of genes involvedin cholesterol and fatty acid synthesis.
TransAM SREBP1 simpliﬁes studying
SREBP1 activation, and detects SREBP-1aand -1c isoforms in nuclear extracts fromhuman, mouse and rat samples (Figure 2).

TransAM SREBP1
Sterol-regulatory element binding
proteins (SREBPs) can be difﬁcult to
study since they are synthesized asinactive precursor proteins bound to
the endoplasmic reticulum and nuclear
envelope, yet once they are activatedby sterol deprivation, the precursor

Widest selection of targets and formats
Active Motif offers the broadest selection of targets, including kits for NFκB,
AP-1, STAT, PPARγ, HIF-1, NFATc1,
C/EBP and many others. Please visit our
website for a complete product listing,and to check for new releases!

Product

FormatCatalog No.

TransAM™ IRF-3 (Human)

1 x 96 rxns
5 x 96 rxns

48396
48896

TransAM™ IRF-3 (Mouse)

1 x 96 rxns
5 x 96 rxns

48496
48996

TransAM™ IRF-7

1 x 96 rxns
5 x 96 rxn

50196
50696

TransAM™ SREBP1

1 x 96 rxns
5 x 96 rxn

50496
50996

Toll Free — 1 877 222 9543

August 2008 • volume 9 • number 2

Magnetic Co-immunoprecipitation

Lower Background Co-IP of Both Nuclear & Whole-cell Complexes
The Universal Magnetic Co-IP Kit includes protein G-coated magnetic beads
that simplify the IP and wash steps while providing lower background, which
improves your Co-IP results. The kit also includes reagents to prepare both
nuclear and whole-cell extracts, so you can study intact protein complexes
whether they were originally bound to DNA or in the cytoplasm.
Protein/protein interactions are often
studied using Co-Immunoprecipitation(Co-IP), in which a single antibody is used
to precipitate an entire protein complex.
Additional antibodies are then used inWestern blot to identify/verify othercomplex members (Figure 1).

Figure 1: Nuclear Co-IP of SRC-1 and ERα.
The Universal Magnetic Co-IP Kit was used to makenuclear extract from MCF-7 cells induced 1 hour with
10 nM Estradiol. IP was performed on 300 µg samples
using 2 µg of SRC-1 pAb, ERα pAb and rabbit IgG (as anegative control). Western blot was then performed
using the ERα pAb on 10 µg Input Extract (Lane 1), SRC-1
IP (Lane 2), ERα IP (Lane 3) and the rabbit IgG IP (Lane 4).

Co-IP cytoplasmic AND nuclear complexes
Co-IP is often used to study cytoplasmic protein complexes. But, traditional
methods are not optimal for studying
DNA-binding proteins because nuclear
complexes are very fragile, causing them
to be disrupted during extraction. For
this reason, in addition to containing
components for preparing whole-cell
extracts, the Universal Magnetic Co-IP
Kit provides nuclear extraction reagentsthat have been optimized to preserve
nuclear protein complexes. The kit’s
Enzymatic Shearing Cocktail uses DNA
digestion to gently release the nuclear
protein complexes from the DNA, sothey are intact and ready for Co-IP.

www.activemotif.com

Simpler procedure, lower background
The Universal Magnetic Co-IP Kit utilizes
protein G-coated magnetic beads, whichsimplify Co-IP by enabling the IP andwash steps to be performed in seconds,
rather than having to use centrifugation.
Because these beads have very low nonspeciﬁc binding, background is reduced
even while using the kit’s low-salt Co-IP/
Wash Buffer, which was designed to help
maintain weaker complexes.
Universal Magnetic Co-IP Kit advantages• Magnetic beads simplify procedureand reduce background• Optimized extraction method maintains nuclear protein complexes• Preserve protein modiﬁcations

Figure 2: Detection of acetylated Histone H3.
HeLa nuclear extracts were made using the Universal
Magnetic Co-IP Kit and a traditional high-salt extraction
protocol, each supplemented with the 1 µM trichostatin
A, a deacetylase inhibitor. Five and ten µg samples ofeach extract were used in Western blot with Histone H3
acetyl rabbit pAb (Cat. No. 39139). Protein was detected
only in samples made using the kit’s gentle nuclear
extraction procedure.

Figure 3: Preservation of acetylation and methylation.
Nuclear extracts were made from HeLa cells treatedwith 500 nM trichostatin A for 20 hours using either
the Universal Magnetic Co-IP Kit (with its deacetylase
inhibitor) or traditional high-salt extraction. Five and tenµg samples of these extracts were used in Western blot
with Histone H3 acetyl pAb (Cat. No. 39139) and Histone
H3 trimethyl Lys4 pAb (Cat. No. 39159). The acetylated
protein was detected only in the sample made using thekit. Methylation was slightly better maintained in thesample made using the kit.

Complete kit for better results
The Universal Magnetic Co-IP Kit has
both nuclear and whole-cell extraction
reagents, so you can perform IP on all
types of protein complexes. The kit also
includes protein G-coated magnetic
beads, a unique Co-IP/Wash Buffer
as well as phosphatase, protease anddeacetylase inhibitors that preserve theintegrity of the proteins and protein
modiﬁcations (Figures 2 & 3). Finally, thekit includes a strong bar magnet, so you
can take advantage of the improved
wash and IP steps enabled by the magnetic beads. This makes the Universal
Magnetic Co-IP Kit a simple, ﬂexible andcomplete solution for getting more fromyour Co-IP. To ﬁnd out more, please give
us a call or visit us on the web atwww.activemotif.com.

Product

FormatCatalog No.

Universal Magnetic Co-IP Kit

25 rxns

54002

9

HAT & HDAC Assays

Rapid, Sensitive Assays for HAT & HDAC Activity and Inhibitor Compounds
Active Motif’s HAT & HDAC Assay Kits are easy-to-use, sensitive assays that can be used to determine the activityof histone acetyltransferases and histone deacetylases in your cell & nuclear extracts, immunoprecipitates andpuriﬁed enzymes, as well as to screen the effects of potential inhibitor compounds. The HAT Assay Kit uses a
ﬂuorescent readout, while HDAC Assay Kits are available in both ﬂuorescent and colorimetric formats.

How does the HAT Assay Kit work?
Assaying HAT activity is easy with this96-well plate format. Simply incubate
your HAT with your choice of theprovided Histone H3 or Histone H4
substrate peptides and acetyl-CoA for
10-30 minutes, then develop. The HAT
Assay Kit uses a thiol-reactive ﬂuorescent dye that reacts with the Co-A-SH
generated by the histone acetyltransferase activity to give a ﬂuorescentreadout. This makes it easy to generate
standard curves with acetyl-CoA or
β-mercaptoethanol, so you can relate
the ﬂuorescence of your HAT to pmol/
min/µg speciﬁc activity.
Active recombinant p300 is provided
as a control for use with your samples;enough is provided so that it could also
be used as a HAT to screen an entire
96-well plate of inhibitors. Anacardic
acid is provided for use as a control, as it
is a potent HAT inhibitor (Figure 1).

10

Assay for HDAC activity or inhibition
With these simple HDAC Assay Kits, you
can easily assay for HDAC activity or you
can screen compounds for their inhibitory effects. The provided deacetylated
assay standard enables you to quantify
HDAC effects with great accuracy.
Figure 1: HAT inhibitor effects on p300 activity.
50 ng p300 were assayed per well with 50 µM acetylCoA and 50 µM histone H3 or H4 peptide substrates in
the absence or presence of 15 µM anacardic acid or
25 µM garcinol, known HAT inhibitor compounds.

How do the HDAC Assay Kits work?
The HDAC Assay Kits utilize a peptide
substrate that contains an acetylated
lysine residue that can be deacetylatedby Class I, II and IV HDAC enzymes. (Class
III HDAC enzymes, or the Sirtuins, require
the addition of the NAD+ cofactor
in the assay.) Once the substrate isdeacetylated, the lysine reacts with theDeveloping Solution and releases either
the chromophore or the ﬂuorophorefrom the substrate to result in either acolorimetric or ﬂuorescent product. The
colorimetric product absorbs maximallyat 405 nm; the ﬂuorescent product canbe read with an excitation wavelength of
360 nm and emission wavelength of
460 nm (Figure 2).

50000

Fluorescence Intensity

Histone acetyltransferases (HAT) are
enzymes that acetylate conserved lysine
amino acids on histones. Generally,
histone acetylation is associated withthe activation of gene expression, as
hyperacetylated chromatin is transcriptionally active. Histone deacetylases(HDAC) remove these acetyl groups
from histones. Their action is opposite
to that of histone acetyltransferases,
as hypoacetylated chromatin is silent.
Because HATs and HDACs are involved
with other proteins in the regulation ofgene expression, their activity is muchstudied, as are compounds that inhibit
HAT and HDAC activity.

Trichostatin A
Untreated

40000
30000
20000
10000
0

10

5

2.5

1.25

0.625

0.312

0.156

0

HeLa Nuclear Extract (µg)

Figure 2: HDAC activity in HeLa cells.
HeLa Nuclear Extracts were assayed at 0 to 10 µg per
well using the fluorescent version of the HDAC Assay
Kit. Untreated extract results are shown with a purple
line, and extracts inhibited with 1 mM Trichostatin A are
shown with a copper line.

Try them today!
Please give us a call or visit us atwww.activemotif.com to get complete
information on our HAT and HDAC
Assays, as well as additional active
histone acetyltransferases and related
assays. Active recombinant p300 and
GCN5 proteins are also available separately for use in other applications.

Product

FormatCatalog No.

HAT Assay Kit (Fluorescent)1 x 96 rxns

56100

Recombinant p300 protein, catalytic domain

5 µg

31205

Recombinant GCN5 protein, active

5 µg

31204

HDAC Assay Kit (Fluorescent)1 x 96 rxns

56200

HDAC Assay Kit (Colorimetric)

1 x 96 rxns

56210

Toll Free — 1 877 222 9543

New Antibodies

August 2008 • volume 9 • number 2

NEW: Antibodies to Study Transcription, Histones and Chromatin Biology
Active Motif offers high-quality antibodies that are manufactured in-house, rigorously tested and validated in theapplications that you need them for.
Validated, specific antibodies
Active Motif specializes in providing
researchers with a wide variety of tools
to study the biology of the nucleus.
If you are studying chromatin biology,transcriptional regulation or cell function, we specialize in making antibodies
to protein targets involved in those
processes. As you can see from our
new product releases, we also specialize in manufacturing antibodies against
histones and histone modiﬁcations,many of which have been validated foruse in chromatin immunoprecipitation(ChIP) and immunoﬂuorescence (IF). We
also offer a wide range of antibodies
against transcription factors, chromatin
modiﬁers and cell-cycle regulators.
Superior characterization
Your choice of antibody supplier iscrucial and can have a dramatic impact
on the success or failure of your experiments. At Active Motif, we are committed to providing researchers with thehighest quality antibodies for studying chromatin and the biology of thenucleus. Every antibody we sell is rigorously tested to ensure that you are notwasting your precious time and researchdollars on antibodies that don’t perform
as advertised. We are not interested injust reselling products from other companies, a very common practice thesedays. We manufacture the antibodies
to histones and histone modiﬁcations
in-house, so we control antibody quality
and performance. Anyone can just SELL
antibodies, but Active Motif actually
makes and characterizes our antibodies
so our antibody performance is guaranteed to work for you.

www.activemotif.com

New Antibody Releases
Cell Biology Antibodies

Applications

Catalog No.

Aurora B Rabbit pAb

IF, WB

39261

INCENP mouse mAb

IF

39259

Lamin A/C Mouse mAb

IF, IP, WB

39287

LAP2 alpha mouse mAb

WB

39267

Applications

Catalog No.

CARM1 Rabbit pAb

WB

39251

Dimethyl-Arginine, asymmetric Rabbit pAb

IF, WB39231

HP1 alpha Rabbit pAb

WB

39295

JARID1C Rabbit pAb

WB

39229

JMJD2D Rabbit pAb

WB

39247

JMJD2F Rabbit pAb

WB

39257

Mi2 beta Rabbit pAb

WB

39289

Histone and Histone Modification Antibodies

Applications

Catalog No.

Histone H2A phospho Ser129 Rabbit pAb (yeast)

ChIP, IF, IP, WB

39271

Histone H2A Rabbit pAb (yeast)

ChIP, WB

39235

Histone H2B Rabbit pAb (yeast)

ChIP, WB

39237

Histone H3 pan-methyl Lys9 Rabbit pAb

IF, WB

39241

Histone H3 monomethyl Lys9 Rabbit pAb

IF, WB

39249

Histone H3 dimethyl Lys9 Rabbit pAb

IF, WB

39239

Histone H3 trimethyl Lys9 Mouse mAb

ChIP, IF, WB

39285

Histone H3 phospho Ser10 Rabbit pAb

IF, WB

39253

Histone H3 dimethyl Lys27 Rabbit pAb

IF, WB

39245

Histone H3 dimethyl Lys36 Rabbit pAb

IF, WB

39255

Histone H3 acetyl Lys56 Rabbit pAb

WB

39281

Histone H3 monomethyl Lys56 Rabbit pAb

WB

39273

Histone H3 dimethyl Lys56 Rabbit pAb

WB

39277

Histone H4 Rabbit pAb

WB

39269

Histone H4 pan-acetyl Rabbit pAb

IF, WB

39243

Histone H4 dimethyl Arg3, symmetric Rabbit pAb

IF, WB

39275

Transcription Factor Antibodies

Applications

Catalog No.

Aiolos Rabbit pAb

IF, IP, WB

39293

Ikaros Rabbit pAb

IF, IP, WB

39291

NFκB p65 Rabbit pAb

WB

39283

RNA pol II CTD phospho Ser5 Rabbit pAb

IF, WB

39233

Epitope Tag Antibodies

Applications

Catalog No.

Myc Tag mouse mAb

WB

39279

Chromatin Modifier Antibodies

11Products for Chromatin &Transcriptional Regulation

THE NEWSLETTER OF ACTIVE MOTIF — August 2008 • volume 9 • number 2•

Recombinant Histones with Site- and Degree-specific Methylated Lysines•

Create Chromatin in vitro with the Chromatin Assembly Kit•

High-throughput ChIP with ChIP-IT™ Express HT•

ChIP Accessory Kits and Reagents•

Double Destabilized Luciferase Provides Better Response & Sensitivity•

Easily Compare CpG Methylation in Various DNA Samples•

Reproducible Bisulfite Conversion for Analysis of Methylated DNA•

Assays for IRF-3 & 7 and SREBP1 Activity•

Lower Background Co-IP of Both Nuclear & Whole-cell Complexes•

Rapid, Sensitive Assays for HAT & HDAC Activity and Inhibitor Compounds•

Antibodies to Study Transcription, Histones and Chromatin Biology

PRSRT STD
US POSTAGEPAID
PERMIT #94
SAN DIEGO, CA

Toll Free: 877 222 9543
Direct: 760 431 1263
Fax: 760 431 1351
Email: sales@activemotif.com
www.activemotif.com

New Transcription, Histones and Chromatin Biology Antibodies•

Rapid, Sensitive Assays for HAT & HDAC•

Lower Background Co-IP of Both Nuclear & Whole-cell Complexes•

Assays for IRF-3 & 7 and SREBP1 Activity•

Reproducible Bisulfite Conversion for Analysis of Methylated DNA•

Compare CpG Methylation in Various DNA Samples•

Double Destabilized Luciferase•

ChIP Accessory Kits and Reagents•

High-throughput ChIP with ChIP-IT™ Express HT•

Create Chromatin in vitro with the Chromatin Assembly Kit•

Histones with Site- and Degree-specific Methylated Lysines•

August 2008 • volume 9 • number 2

THE NEWSLETTER OF ACTIVE MOTIF

Products for Chromatin & Transcriptional Regulation