THE NEWSLETTER OF ACTIVE MOTIF — March 2009 • volume 10 • number 1

High-quality Antibodies and Reagents for Epigenetics,
Chromatin Biology & Cell Signaling

IN
THIS
ISSUE

2 Retroviruses, Chromatin Structure andTranscriptional Regulation

7 Rigorous Testing Ensures Consistent
Antibody Performance

4 NEW: HeLa Acid Extracts for
Epigenetic Studies

8 Reconstitute Chromatin Quickly and Easily
with the Chromatin Assembly Kit

4 Histones Containing Site- and Degreespeciﬁc Methylated Lysine Residues

8 Easily Purify Core Histones from Any Cell
Culture or Tissue Sample

5 Bisulﬁte Conversion for Accurate Analysis
of Methylated DNA

9 Assays to Measure HAT & HDAC Activity

5 Isolation of CpG-Methylated DNA Using
Recombinant Methyl-binding Protein
6 Upgrade your Experiments using Magnetic
Bead-based ChIP-IT™ Express Kits
7 Improve Your ChIP Experiments with
ChIP Accessory Kits and Reagents

10 In-cell ELISAs for More Quantitative
Monitoring of Phosphorylation
11 Simpliﬁed Study of SUMOylation Effects
with SUMOlink™
11 Accurately Measure DNA Repair
Protein Activity

Review Article

Retroviruses, Chromatin Structure and Transcriptional Regulation
Retroviral DNA is integrated in its host genome, which allows the virus toescape from the host’s immune system. Therefore, chromatin structure plays
an important role in the transcriptional regulation of retroviral genes.
During retroviral infection of aeukaryotic cell, the integration of viral
DNA into the cellular genome is an
important step for the viral cycle. This
allows the provirus to survive in the hostcell and, with limited viral transcription,
escape detection by the host immune
system. Once integrated, the provirus
is organized into chromatin, along withall cellular genes, and is transcribed by
the host RNA polymerase II (RNA pol II).
Transcription from eukaryotic promoters, including retroviral promoters, isregulated by different cellular mechanisms. Recruitment of transcription
factors, chromatin structure, histone
modiﬁcations and DNA methylation play
important roles in this process.
Chromatin, the material into which
genomic DNA is packaged in eukaryotes,
is a very dynamic structure. The smallest
subunit of chromatin is the nucleosome,147 base pairs of DNA wrapped aroundan octamer of core histone proteins.
Chromatin is subject to a variety ofchemical modiﬁcations, including thepost-translational modiﬁcation of
the histones and the methylation ofcytosines in the DNA. Reported histone
modiﬁcations include acetylation,
methylation, phosphorylation, ubiquitylation, glycosylation, ADP-ribosylation,
carbonylation and SUMOylation. Many
modiﬁcations can and do inﬂuence others, and many are positively or negatively correlated with speciﬁc transcriptional
states and the speciﬁc organization ofrepressive or open chromatin. Some
modiﬁcations serve as signals for thebinding of speciﬁc proteins, referred tocollectively as the “histone code”.

2

After integration of the viral DNA into
the host genome, the provirus can betranscriptionally active or inactive (latent). For example, cells latently infectedby Human Immunodeﬁciency Virus type
1 (HIV-1) serve as “reservoirs” of virus andare a permanent source of virus reactivation. Latency is a viral strategy to avoid
the host immune response, allowing
survival of the virus. The persistenceof these latently HIV-infected cellularreservoirs, despite prolonged treatmentgroup to cytosines is carried out by
the DNA methyltransferase enzymes(DNMT). There are at least two generalmechanisms by which DNA methylation
inhibits gene expression: ﬁrst, modiﬁcation of cytosine bases can inhibit theassociation of some DNA-binding factors
with their cognate DNA recognition
sequences;3 and second, proteins thatrecognize methyl-CpGs can elicit the
repressive potential of methylated DNA(reviewed in 4). The second mechanism ispredominant and is generally associatedwith histone deacetylation, rendering
the conformation of chromatin inaccessible to the transcriptional machinery.
Additionally, there is a signiﬁcant body

An inverse correlation between gene expression and CpG methylation in the
5´LTR of retroviral genomes has been demonstrated in HTLV-1, in
Moloney Murine Leukemia Virus (Mo-MuLV) and in Rous Sarcoma virus.
with highly effective retroviral therapy,
represents the major obstacle to virus
eradication. Bovine Leukemia Virus (BLV)and Human T-Lymphotropic Virus type 1(HTLV-1) infections are characterized byviral latency in the large majority of infected cells and by the absence of virus
circulating in the blood. These features
are thought to be due to the repressionof viral gene expression in vivo.1, 2
In this review, we will focus on the roleof some chromatin modiﬁcations in
the initiation and maintenance of virallatency within the host cells.
DNA methylation
Inhibition of gene expression and a
repressive chromatin state are often
associated with DNA methylation,
an epigenetic modiﬁcation of DNA
that occurs on cytosines within CpG
dinucleotides. Addition of the methylof evidence linking DNA methylation
with histone methylation, indicating that
one might reinforce the other to cooperatively silence gene expression.37-39
Methylation of retroviral promoter
and enhancer sequences located in
the 5´LTR (Long Terminal Repeat) is amechanism of epigenetic silencing ofprovirus transcription, which allows
the virus to become latent and avoid
detection by the host immune system.5-7
An inverse correlation between gene
expression and CpG methylation in the
5´LTR of retroviral genomes has indeed
been indeed demonstrated in HTLV-1,7-9
in Moloney Murine Leukemia Virus (MoMuLV)6, 10 and in Rous Sarcoma virus.11
The role of HIV-1 promoter methylationin viral latency is quite controversial,
however. CpG methylation within the
HIV-1 promoter inhibits transcription ofin vitro-methylated plasmids transfected

Toll Free — 1 877 222 9543

March 2009 • volume 10 • number 1
into cells,12-14 and it was suggested as amechanism to maintain HIV-1 latency insome infected cell lines.15, 16 In contrast tothese studies, Pion et al. showed using
bisulﬁte sequencing, that transcriptional
repression of HIV-1 is not associated withmethylation of the 5´LTR.17
Histone acetylation
One factor inﬂuencing the modulationof chromatin structure is the reversible
acetylation of conserved lysine residues
on histone proteins. The acetylation
reaction results in the transfer of theacetyl group from acetyl-coA to the
ε-amino group of the lysine residue,
neutralizing its positive charge. Steadystate levels of histone acetylation result
from a balance between the activityof two families of antagonistic enzymes: histone deacetylases (HDACs)
and histone acetyltransferases (HATs),
which respectively remove or add acetylgroups to histones.
Histone acetylation is an importantmechanism implicated in the regulationof retroviral promoters. Several studies have reported the regulation of BLV
and HTLV-1 transcription by histone
acetylation. Many groups have observed
the strong effect of HDAC inhibitors on
the BLV gene expression in vitro and invivo.18-21 They have also shown a role for
the HATs CBP and p300 in the transcriptional activation of BLV and HTLV-1.22-25
Recruitment of HDAC1, HDAC2 or
HDAC3 to the HTLV-1 promoter has been
observed and linked to repression of theviral promoter.26, 27 Other retroviruses
including HIV-1 (review in 28), the Rous
Sarcoma virus29 or MoMuLV30, 31 are also
regulated by histone acetylation. For
example, it has been demonstrated bychromatin mapping experiments thata nucleosome positioned immediately
downstream of the transcription startsite is remodeled upon the activation of
the HIV-1 promoter in response to HDAC

www.activemotif.com

inhibitors.32 Lorincz et al. have demonstrated an inverse correlation between
acetylation of histone H3 and proviralmethylation density and transcriptional
repression of MoMuLV.30
Histone Methylation
The methylation of histones can occur
on arginine (R) and lysine (K) residues.
Methylation at many residues of all
the core histones has been observed(reviewed in 33). Histone lysine methyltransferases (HMTs) contain a catalytic
SET domain, utilizing S-adenosyl-Lmethionine (SAM) as a cofactor. Somesites of histone methylation are associated with transcriptional activation (e.g.
H3 K4), whereas others (e.g. H3 K9, H3
K27) are associated with transcriptional
repression and heterochromatin formation. The arginine methyltransferases are
responsible for the transfer of methylgroups from SAM to the guanidinium

Histone acetylation is also an
important mechanism in regulation ofretroviral promoters.
group of arginine. There are several
enzymes which methylate histones atarginine residues, PRMT1 and CARM1/
PRMT4 being the most widely studied ofthese. Generally, methylation of histones
on arginine residues is associated withtranscriptional activation of genes.
A few things are known about histone
methylation within the BLV promoter.
Merimi et al. demonstrated a change inlysine methylation leading to BLV activation after a treatment of the infected
cells with a combination of trichostatin
A (an HDAC inhibitor) and 5-azacytidine(a DNA methylation inhibitor).34 An
increase of H3 K4 methylation and adecrease of H3 K9 methylation accompanied a decrease of HDAC1 and mSin3
recruitment after treatment of the

Review Article
cells.34 In the case of HTLV-1, two histone
methyltransferases seem to be involvedin the transcriptional regulation of theviral promoter.35, 36 ChIP assays demonstrated the recruitment of CARM1 andSuv39H1 in vivo at the promoter region,
together with the viral transcriptional
activator protein, Tax. This recruitment
is associated with strong H3 methylation
on different arginine residues and withacetylation of H3 K9 in cells containing asingle active integrated copy.
All these studies show the importanceof epigenetic modiﬁcations in the transcriptional regulation of retroviruses. It
is certain that similar future research willaid in treatment of viral diseases.
References

1. N. Gillet et al., (2007) Retrovirology 4:18.
2. J. Yasunaga, M. Matsuoka, (2007) Cancer Control 14:133.
3. F. Watt, P. L. Molloy, (1988) Genes Dev 2:1136.
4. T. Latham, N. Gilbert, B. Ramsahoye, (2008) Cell Tissue
Res 331:31.
5. R. Pearson et al.,(2008) J Virol 82:12291.
6. K. Harbers, A. Schnieke, H. Stuhlmann, D. Jahner, R.
Jaenisch, (1981) PNAS 78:7609.
7. T. Koiwa et al., (2002) J Virol 76:9389.
8. D. Saggioro, M. Panozzo, L. Chieco-Bianchi, (1990) Cancer
Res 50:4968.
9. Y. Taniguchi et al., (2005) Retrovirology 2:64.
10. R. C. Hoeben, A. A. Migchielsen, R. C. van der Jagt, H. van
Ormondt, A. J. van der Eb, (1991) J Virol 65:904.
11. J. Hejnar et al., (1999) Virology 255:171.
12. D. P. Bednarik, J. A. Cook, P. M. Pitha, (1990) EMBO J 9:1157.
13. K. A. Gutekunst, F. Kashanchi, J. N. Brady, D. P. Bednarik,(1993) J Acquir Immune Defic Syndr 6:541.
14. K. Schulze-Forster, F. Gotz, H. Wagner, H. Kroger, D.
Simon, (1990) Biochem Biophys Res Commun 168:141.
15. M. K. Singh, C. D. Pauza, (1992) Virology 188:451.
16. T. Ishida, A. Hamano, T. Koiwa, T. Watanabe, (2006)
Retrovirology 3:69.
17. M. Pion et al., (2003) J Virol 77:4025.
18. C. Merezak et al., (2002) J Virol 76:5034.
19. A. Achachi et al., (2005) PNAS 102:10309.
20. C. Calomme et al., (2004) J Virol 78:13848.
21. T. L. Nguyen et al., (2004) J Biol Chem 279:35025.
22. T. L. Nguyen et al., (2007) J Biol Chem 282:20854.
23. H. Lu et al., (2002) Mol Cell Biol 22:4450.
24. I. Clerc et al., (2008) J Biol Chem 283:23903.
25. N. Sharma, J. K. Nyborg, (2008) PNAS USA 105:7959.
26. R. Villanueva et al., (2006) Oncol Rep 16:581.
27. T. Ego, Y. Ariumi, K. Shimotohno, (2002) Oncogene 21:7241.
28. V. Quivy, S. De Walque, C. Van Lint, (2007) Subcell
Biochem 41:371.
29. E. Espinos, A. Le Van Thai, C. Pomies, M. J. Weber, (1999)
Mol Cell Biol 19:3474.
30. M. C. Lorincz, D. Schubeler, M. Groudine, (2001) Mol Cell
Biol 21:7913.
31. R. Appanah, D. R. Dickerson, P. Goyal, M. Groudine, M. C.
Lorincz, (2007) PLoS Genet 3, e27.
32. C. Van Lint, S. Emiliani, M. Ott, E. Verdin, (1996) EMBO J
15:1112.
33. P. K. Lo, S. Sukumar, (2008) Pharmacogenomics 9:1879.
34. M. Merimi et al., (2007) J Virol 81:5929.
35. S. J. Jeong et al., (2006) J Virol 80:10036.
36. Y. Zhang, D. Reinberg, (2001) Genes Dev 15:2343.
37. L. M. Johnson et al., (2007) Curr Biol 17:379.
38. S. Epsztejn-Litman et al, (2008) Nat Struct Mol Biol
15:1176.
39. J. Wang et al, (2009) Nat Genet 41:125.

3

Histone Modifications

NEW: HeLa Acid Extracts for Epigenetic Studies
Active Motif’s commitment to providing researchers with quality products to complement their chromatin andepigenetic studies continues with the introduction of HeLa acid extracts. Active Motif is now offering HeLa acidextracts that are either untreated, or treated with chemicals known to affect epigenetic events. These acid extracts
also serve as controls for many of the histone modiﬁcation antibodies that Active Motif sells. To learn more about
either the acid extracts, or associated histone modiﬁcation antibodies, please visit www.activemotif.com.
17 kDaProduct

FormatCatalog No.

HeLa acid extract

100 µg

36200

HeLa acid extract (Paclitaxel treated)

100 µg

36201

HeLa acid extract (Sodium butyrate treated)

100 µg

36202

HeLa acid extract (Etoposide treated)

100 µg

36203

HeLa acid extract (Anacardic acid treated)

100 µg

36204

1

2

Figure 1: Western blot of HeLa acid extracts.
10 µg of HeLa acid extract that was either untreated(lane 1) or treated with 1 µg/ml paclitaxel for 20 hours(lane 2) was used in a Western blot with Histone H3
phospho Ser28 pAb (Cat. No. 39149). Paclitaxel is a
mitotic inhibitor that arrests cells as they enter mitosis.
Histone H3 serine 28 serves as a mitotic marker as it ishighly phosphorylated during mitosis.

Histones Containing Site- and Degree-speciﬁc Methylated Lysine Residues
Histones are subject to a variety of modiﬁcations including acetylation, phosphorylation, methylation, ubiquitination
and ADP ribosylation. The type and number of modiﬁcations determine chromatin structure and the extent of geneactivation or silencing. Generally, methylation on histones H3K4, H3K36 and H3K79 is associated with transcriptional
activation while methylation on H3K9, H3K27 and H4K20 is associated with transcriptional repression. To better
investigate how methylation patterns impact regulatory processes, assays will require histones with speciﬁcmethylation states, such as the site- and degree-speciﬁc methylated lysine histones offered by Active Motif.
How is the methylation state made?
Active Motif now offers recombinanthistones with site- and degree- speciﬁc lysine methylation. Recombinant
methylated histones are created using apatented approach involving a chemical alkylation reaction that introduces
an analog of methyl lysine. This speciﬁc
treatment allows the site and degree ofmethylation to be precisely controlled.
All recombinant histone modiﬁcations
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, see page 8). One µg ofassembled chromatin was digested for 2 minutes(Lane 2) and 4 minutes (Lane 3). Lane 1 is 100 bp marker.

are conﬁrmed by ESI-TOF mass spectrometry and dot blot or immunoblot.
Because the methylation state closely
mimics natural methylation, theserecombinant proteins are perfect forstudying the effects of speciﬁc lysine
methylations on nucleosome remodeling, the binding of associated chromatinproteins, or for use as building blocks forin vitro chromatin assays (Figure 1).1
1

Lu, X. et al. (2008) Nat. Str. & Mol. Biol. doi:10.1038/nsmb.1489

Product

FormatRecombinant Histone H3 (C110A) or Histone H4

50 µg

Recombinant Histone H3 mono-, di-, or tri-methyl Lys4

50 µg

Recombinant Histone H3 mono-, di-, or tri-methyl Lys9

50 µg

Recombinant Histone H3 mono-, di-, or tri-methyl Lys27

50 µg

Recombinant Histone H3 mono-, di-, or tri-methyl Lys79

50 µg

Recombinant Histone H4 mono-, di-, or tri-methyl Lys20

50 µg

For additional details and ordering information, visit www.activemotif.com/recomhistones.

4

Toll Free — 1 877 222 9543

March 2009 • volume 10 • number 1

DNA Methylation

Analyzing DNA Methylation Aids the Study of Development and Disease
DNA methylation is an epigenetic event that affects cell function by altering gene expression. DNA methylation is
involved in embryonic development and cell cycle regulation, while aberrant methylation is prevalent in many humancancers. During methylation, methyl groups are transferred from the cofactor, S-adenosyl-L-methionine, to the ﬁfthcarbon of cytosine in a CpG dinucleotide. This reaction is catalyzed by one of the DNA methyltransferase enzymes.
Due to the importance of methylation in development and disease, analyzing DNA methylation is necessary forunderstanding gene expression. Active Motif offers two techniques to aid in DNA methylation analysis.

Bisulﬁte Conversion for Accurate Analysis of Methylated DNA
What is bisulﬁte conversion?
One method to analyze DNA methylation is through bisulﬁte conversion.
Bisulﬁte modiﬁcation involves theconversion of unmethylated cytosines
into uracils, while methylated cytosines
remain unchanged. The DNA is thenampliﬁed by PCR and analyzed bysequencing or restriction digest. A
comparison of the sequences of converted and untreated DNA will reveal
the methylation proﬁle of the sample.

The MethylDetector™ advantage
Active Motif’s MethylDetector™ Bisulﬁte
Kit simpliﬁes analysis of DNA methylation by providing optimized reagents for
performing DNA conversion, timesaving
DNA puriﬁcation columns and positive
control PCR primers that are speciﬁc forbisulﬁte-converted DNA. This allows you

to conﬁrm the success of the conversion procedure before spending extra
time and money on sequencing, or otheranalysis methods.
For complete product details, please
visit www.activemotif.com.

Product

FormatCatalog No.

MethylDetector™

50 rxns

55001

Isolation of CpG-Methylated DNA Using Recombinant Methyl-binding Protein
Why isolate methylated CpG islands?
A second method for the analysis of
DNA methylation is the isolation of
CpG-methylated DNA. CpG islands are
small regions of the DNA in which the
CpG dinucleotide frequency is higher
than would normally be expected. WhileCpG islands are only found in approximately 1% of the genome, more than
60% of human promoters contain CpG
islands. Studying CpG island methylation
is of considerable importance because
CpG islands are normally not methylated. If a CpG island within a promoter
becomes methylated, the gene associated with the promoter is permanently
silenced, and this silencing can be transmitted through mitosis. This means thatCpG island methylation is an epigenetic
means of inheritance.

www.activemotif.com

How does MethylCollector™ work?
Active Motif’s MethylCollector™ Kit
utilizes the recombinant Methyl-binding
protein (MBD2b) to capture CpG-methylated DNA from enzymatically digested
or sonicated cell or tissue samples. The
kits’ optimized buffers remove DNA
fragments that have little or no methylation, eluting puriﬁed, methylated DNA
that is ready for PCR analysis of the locus of interest (Figure 1). Positive control
DNA and PCR primers are also included
to help ensure success.
Learn more at www.activemotif.com.

0

1

2

MBD2b (µg)
0
1
1

3

4

5

Input

6

7

8

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.

Product

FormatCatalog No.

MethylCollector™

25 rxns

55002

Fully Methylated Jurkat DNA

10 µg

55003

5

Magnetic-bead Based Chromatin IP

Upgrade your Experiments using Magnetic Bead-based ChIP-IT™ Express Kits
Active Motif’s line of ChIP-IT™ Express Kits and reagents make chromatin IP faster and more consistent. Kits areavailable in either sonication or enzymatic shearing formats, with or without positive and negative controls. ChIP-IT
Express Kits employ protein G-coated magnetic beads, making it possible to simplify the ChIP protocol, which
reduces your time and effort (Figure 1). We have also extended our ChIP-IT Express product line to include two new
kits: ChIP-IT™ Express HT, for high-throughput ChIP, and Re-ChIP-IT™, making it easy to perform sequential IP.
ChIP-IT magnetic bead advantages• Faster procedure – magnetic sortingis much faster than centrifugation• No pre-blocking needed –magnetic beads are inert and exhibit
low binding of proteins• Less effort required – Our kits
are compatible with multi-channel
pipettors to streamline wash steps• No need for DNA purification –our specialized ChIP Elution Buffer
eliminates the need for expensive
DNA puriﬁcation columns• Perform ChIP with fewer cells –routinely perform chromatin IP
with 100,000 cells or fewer
The Innovation Leader in ChIP products
Active Motif was the ﬁrst company tointroduce a ChIP kit employing magnetic
beads. We have recently strengthened
our position as the leader in developing
innovative chromatin IP kits with therelease of two new kits that will allow
you to expand the utility of the ChIP
technique: ChIP-IT Express HT, allowing

Figure 1: PCR of chromatin IP performed on 100,000 cells.
Typically, ChIP requires 2 million cells per reaction. With
ChIP-IT Express, it is possible to perform successful ChIP
with 100,000 cells or less. In the gel above, ChIP was
performed in duplicate on chromatin isolated from
100,000 HeLa cells using the Negative Control IgG, RNA
pol II antibody and GAPDH PCR primers supplied in the
ChIP-IT Control Kit – Human (Cat. No. 53010, see page 7).

6

the processing of up to 96 ChIP reactions
simultaneously, and Re-ChIP-IT, making
it easy for you to perform sequential
chromatin IP.
High-throughput ChIP a reality
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 bead-based
protocol of ChIP-IT Express with a highthroughput, 96-well microplate-basedformat (Figure 2). 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 as with the ChIP-IT Control Kits.
Identify protein co-localization in vivousing sequential chromatin IP
When performing ChIP experiments,
it is often useful to prove that two
different proteins or histone modiﬁcations are present at the same site
in the genome. Or, you may want todetermine if a protein coincides witha speciﬁc histone modiﬁcation at thesame regulatory element. Sequential
ChIP (also called Re-ChIP) is a relatively
new technique in which sequential

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

chromatin immunoprecipitation reactions are performed using two different
antibodies, enabling you to assay for thesimultaneous presence of two proteinsor distinct histone modiﬁcations at
the same genomic region of interest.
Performing sequential chromatin IP wastechnically challenging and difﬁcult,until now. Active Motif’s new Re-ChIP-IT
Kit makes it easy to perform sequential
ChIP, so you can localize two different
proteins or histone modiﬁcations to thesame genomic locus.
Are you ready to upgrade your ChIP
experiments? Then please visit us atwww.activemotif.com/chipitexpress
to learn more about the ChIP-IT
Express line of ChIP kits, reagentsand ChIP-validated antibodies.

Product

FormatCatalog No.

ChIP-IT™ Express HT

96 rxns

53018

ChIP-IT™ Express

25 rxns

53008

Re-ChIP-IT™

25 rxns

53016

ChIP-IT™ Express Enzymatic

25 rxns

53009

ChIP-IT™ Protein G Magnetic Beads

25 rxns

53014

Toll Free — 1 877 222 9543

March 2009 • volume 10 • number 1

Chromatin IP Accessories & Quality Antibodies

Improve Your ChIP Experiments with ChIP Accessory Kits and Reagents
Active Motif offers a broad range of reagents and accessory kits to complement its ChIP-IT™ and ChIP-IT™ Express lineof ChIP kits. These products will help you troubleshoot your ChIP experiments and make them more reproducible.
Superior ChIP-IT magnetic beads
When you use Active Motif’s ChIP-IT
Protein G Magnetic Beads, the protein G
is directly conjugated to a low proteinbinding magnetic bead, so pre-blockingof the beads is not required. ChIP-IT
Protein G Magnetic Beads are validated
for use in chromatin IP, and ready to use.
Simpliﬁed chromatin shearing
Employing sonication to shear chromatin
for ChIP can be tedious and difﬁcult toperform reproducibly. To eliminate theproblems associated with sonication,
Active Motif has developed a morerobust and user-friendly method thatuses our proprietary Enzymatic Shearing
Cocktail to quickly shear chromatin. As
enzymatic shearing is solely time andtemperature dependent, inconsistencies
associated with sonication are eliminated, helping improve your ChIP results.

ChIP-IT Control Kits
Chromatin IP is a complex technique,so it can be difﬁcult to determine if theexperiment worked. To make your ChIP
experiments easier to troubleshoot,Active Motif sells ChIP-IT Control Kits
for human, mouse and rat cells withpositive and negative control antibodies
and positive control PCR primers.

Ready-to-ChIP Chromatin
For your convenience, Active Motif
offers Ready-to-ChIP Chromatin from avariety of common human and mouse
cell lines. Ready-to-ChIP Chromatin has
been optimally sheared by sonicationand validated in ChIP. As a result, you canmore easily validate your own antibodies
and primer sets.

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

Ready-to-ChIP Hep G2 Chromatin

10 rxns

53019

Ready-to-ChIP K-562 Chromatin

10 rxns

53020

Ready-to-ChIP NIH/3T3 Chromatin

10 rxns

53021

ChIP-IT™ Shearing Kit

10 rxns

53002

Enzymatic Shearing Kit

10 rxns

53005

Rigorous Testing Ensures Consistent Antibody Performance
Active Motif is committed to providing the highest quality antibodies for studying chromatin and the biology of thenucleus. We specialize in manufacturing antibodies against histones, histone modiﬁcations and chromatin proteins,
many of which have been validated for use in ChIP and immunoﬂuorescence. Each antibody we make is rigorously
tested to ensure you won’t waste your precious time and research dollars on antibodies that don’t perform as speciﬁed.
Our Antibody Commitment• Quality first – we’d rather fail our
project than sacriﬁce quality• Highly characterized – all of ourantibodies are stringently tested
under multiple conditions• Controlled process – we manufacture and test our own antibodies• Consistent – we go to great lengths
to minimize lot-to-lot variability

Expert manufacturers of:• Histone and Histone Modiﬁcation
Antibodies• Transcription Factor andCell Signaling Antibodies• Antibodies to Chromatin Modifying
Proteins• ChIP-validated Antibodies

To ﬁnd the ones you need from the over 350 high-quality antibodies offered by Active
Motif, use the convenient search tools found at www.activemotif.com/abs.

www.activemotif.com

Immunoﬂuorescence with Active Motif antibodies.
HeLa cells stained with Aurora B pAb (Cat. No. 39261).
Aurora B (stained in red, but appears yellow) is visualized
in the spindle midzone of the mitotic cell on the right.
Green: alpha Tubulin mAb (Cat. No. 39527). Blue: DAPI.

7

Chromatin Assembly & Histone Purification

Reconstitute Chromatin Quickly and Easily with the Chromatin Assembly Kit
The 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 the regulation of your target gene.
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 regularly spaced
nucleosomes is made by adding the supplied components to 1 µg of your linear
or supercoiled DNA, then incubating for
4 hours. Micrococcal nuclease digestionof the resulting chromatin conﬁrms theordered spacing of nucleosomes (Figure 1).

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
Try in vivo assembly of chromatin today
The Chromatin Assembly Kit makes it
easy for you to assemble chromatin on

M

1

2

3

4

Figure 1: Enzymatic digestion of assembled chromatin.
Limited micrococcal nuclease digestion was carried out
on chromatin assembled using 1 µg samples of circular
DNA (Lanes 1 & 2) or linear DNA (Lanes 3 & 4), resulting
in regularly spaced nucleosomes.

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

Easily Purify Core Histones from Any Cell Culture or Tissue Sample
Now you can easily purify histones and further separate the fractions of core histones from any cell culture or tissue
sample while maintaining post-translational modiﬁcations like acetylation, methylation and phosphorylation. Active
Motif has two versions of our Histone Puriﬁcation Kit to meet your experimental needs.
How does it work?
Our unique kits utilize a patented method and proprietary binding columns
and elution buffers to isolate very pure
fractions of histones. Using the Histone
Puriﬁcation Mini Kit, core histones canbe puriﬁed from as few as 8 x 105 cultured cells or tissue samples as a single
population containing H2A, H2B, H3 andH4. With the original Histone Puriﬁcation
Kit, several milligrams of core histones
can be puriﬁed as one total population
or fractionated into separate pools of
H2A/H2B dimers and H3/H4 tetramers(Figure 1). Both kits utilize a convenient8

spin-column format that greatly reduces
the processing time required.

MW
98
64
50

Histone Puriﬁcation Kit advantages• Rapid and convenient protocol
reduces effort• Multiple kits to meet your histone
puriﬁcation needs• Post-translational modiﬁcations
remain intact• Puriﬁed histones will improve yourdownstream assays

36
30

H3

16

H2B
H2A

H4
6
4

Figure 1: Separate H2A/H2B and H3/H4 fractions.
H2A/H2B and H3/H4 fractions isolated from HeLa cells.

Product

FormatCatalog No.

Histone Puriﬁcation Mini Kit

20 rxns

40026

Histone Puriﬁcation Kit

10 rxns

40025

Toll Free — 1 877 222 9543

March 2009 • volume 10 • number 1

HAT & HDAC Assays

Assays to Measure HAT & HDAC Activity
Histone acetyltransferases (HATs) are enzymes that acetylate conserved lysine amino acids on histones by transferring
an acetyl group from acetyl coenzyme A (acetyl-CoA). Generally, histone acetylation is associated with activation ofgene expression, but it is also involved in DNA replication, histone deposition and DNA repair. Histone deacetylases(HDACs) remove the acetyl groups from histones creating hypoacetylated chromatin, which is transcriptionally silent.
Because of the importance of histone acetylation in chromatin function, HATs and HDACs have major roles in thecontrol of cell fate and compounds that activate or inhibit their activity are of great interest. Active Motif offers
both HAT and HDAC Assay Kits that make it easy to accurately determine the activity of histone acetyltransferases
and histone deacetylases in your sample, as well as the effect of inhibitor compounds.

Fluorescence Intensity

50000

Trichostatin A
Untreated

40000
30000
20000
10000

0

10

5

2.5

1.25

0.625

0.312

0.156

35000

Histone H3
Histone H4

30000

Figure 2: Effect of anacardic
acid on p300 HAT activity.
50 ng of recombinant p300
protein was assayed with 50 µM
acetyl-CoA and 50 µM Histone
H3 or H4 peptide substrates in
the absence or presence of
15 µM anacardic acid, a known
HAT inhibitor.

25000
20000
15000
10000
5000
0
Backgroundp300

Anacardic Acid

p300 Activity

How does the HAT Assay Kit work?
The HAT Assay Kit is a quick and simple
assay that is performed in a 96-wellplate. The kit includes Histone H3 andHistone H4 substrate peptides that are
combined with your sample in the presence of acetyl-CoA. Activity is measured
within 10-30 minutes by a ﬂuorescentreadout of the thiol-reactive dye with
the Co-A-SH that is a by-product of the
HAT reaction. Active recombinant p300
protein, which is capable of acetylating

all four core histones, is included as apositive control. Anacardic acid, a potent
HAT inhibitor, is also included for use asa negative control (Figure 2).
Order today!
Please give us a call or visit us atwww.activemotif.com to get additional
information on our HAT and HDAC
Assays. Active recombinant p300 and
GCN5 proteins are also available separately for use in other applications.

0

HeLa Nuclear Extract (µg)

Figure 1: 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, while extracts inhibited by 1 mM Trichostatin A are
shown with a copper line.

www.activemotif.com

40000

Arbitrary Fluorescence Units (AFU)

How do the HDAC Assay Kits work?
The HDAC Assay Kits are available inboth Fluorescent and Colorimetricformats. They utilize an acetylated
lysine peptide substrate that can bedeacetylated by Class I, II and IV HDAC
enzymes. (Class III HDAC enzymes, or
the Sirtuins, require the addition of the
NAD+ cofactor into the assay). Oncethe substrate is deacetylated, the lysine
reacts with the Developing Solution
and releases either a chromophore or
ﬂuorophore. Trichostatin A is provided
for use as an HDAC inhibitor (Figure 1).

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

9

Phospho-Protein Detection

In-cell ELISAs for More Quantitative Monitoring of Phosphorylation

FACE advantages• Cell-based – no extraction, gelsor blotting needed• Accurate – ﬁxation prevents
additional protein modiﬁcations• Fast – 5-hour protocol, with just
2 hours of hands-on time
• Grow cells in 96-well plates – saveon reagents• Economical – total and phosphospeciﬁc antibodies provided for2 x 96 wells• Semi-quantitative results – normalize to total protein and cell number

treated to induce phosphorylation of
the protein of interest. The cells are
rapidly ﬁxed with formaldehyde topreserve the phosphorylation state,
and then each well is incubated with a
primary antibody speciﬁc for either the
total protein or its phosphorylated form.
Subsequent incubation with a labeledsecondary provides a colorimetric or
chemiluminescent readout that is quantitative and reproducible (Figures 1 & 2).
Data is easily normalized to cell number
using the provided Crystal Violet Dye.
A variety of kits to choose from
FACE Kits are available for over 20 different targets (Table 1). The Suspension
Cell FACE module was designed to workwith all FACE Kits; it improves results
when working with suspension cells by

The “in-cell” method
Fast Activated Cell-based ELISAs (FACE™)
are easy to use and require just
2 hours of hands-on time. Cells aregrown in 96-well cell culture plates and

Add anti-IgG
HRP conjugate

Add primary
antibody
3 hrs.

1 hr.

Cells arefixed following
stimulation

P

P

P

providing 96-well ﬁlter plates that makeit easier to perform washing & liquidhandling steps. And, with FACE Maker
Kits, you can use your own primary andsecondary antibodies to detect any
target or modiﬁcation state of interest.
Simplify your phospho-assays today!
For complete information on the FACE
product line, please give us a call or visit
us at www.activemotif.com/face.

1

1

Cells cultured in
96-well plate

Figure 1: Flow chart of the FACE process.
Cells are grown, stimulated and fixed in the same 96-well plate. Addition of primary and secondary antibodies detects total
protein as well as the phosphorylated form of the protein.

3

4

2

3

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

A

4
43 kDaAdd developing
and stop solution

Stimulatecells

2

43 kDaOD450 nmActive Motif’s Fast Activated Cell-based ELISA (FACE™) Kits are a simple,
sensitive, cell-based method for detecting protein phosphorylation directlyin the cell, without the trouble of making cell extracts or running gels.

B

Phospho p38
Total p38

C

0

0.1
1
10
Anisomycin induction (µg/ml)

Figure 2: Phospho and total p38 MAPK assays.
Macrophage 4/4 cells were grown in 10 cm dishes to
80% confluency, serum-starved for 16 hours and stimulated with anisomycin for 15 minutes. Cell lysates weremade and Western blots performed using phospho- (A)and total-p38 antibodies (B). For FACE, 4/4 cells weregrown in 96-well plates, stimulated as above, fixed andthen assayed in triplicate using the FACE p38 Kit (C). Data
were corrected for cell number through use of the kit’s
Crystal Violet Dye. Western blot data provided courtesyof Dr. Henri H. Versteeg and Dr. Maikel P. Peppelenbosch.

Table 1: The FACE™ Product Line
FACE™ AKT (S473)

FACE™ ATF-2 (T71)

FACE™ Bad (S112)

FACE™ c-Jun (S63)

FACE™ c-Jun (S73)

FACE™ c-Src (Y418)

FACE™ EGFR (Y845)

FACE™ EGFR (Y992)

FACE™ EGFR (Y1173)

FACE™ ErbB-2 (Y877)

FACE™ ErbB-2 (Y1248)

FACE™ ERK1/2 (T202/Y204& T185/Y187)

FACE™ FAK (Y397)

FACE™ FKHR (FOXO1) (T24) FACE™ GSK3β (S9)

FACE™ HSP27 (S82)

FACE™ JAK1 (Y1022/Y1023) FACE™ JNK (T183/Y185)

FACE™ PI3 Kinase p85

FACE™ STAT2 (Y869)

FACE™ MEK1/2 (S217/S221) FACE™ NFκB Proﬁler(S468 & S536)

FACE™ p38 (T180/Y182)

FACE™ STAT6 (Y641)

Suspension Cell FACE™

10

FACE™ Maker

FACE™ STAT4 (Y693)

Toll Free — 1 877 222 9543

March 2009 • volume 10 • number 1

SUMOylation & DNA Repair ELISAs

Simpliﬁed Study of SUMOylation Effects with SUMOlink™
SUMOlink™ Kits are a simple, effective method for generating SUMOylated
proteins in vitro. These fast, efﬁcient and robust assays contain all necessary
reagents for SUMOylation of target proteins, and include positive andnegative controls that help to ensure your success.

MW

1

2

3

1

2

3

98
64
50
36

The SUMOlink method
SUMOlink™ Kits enable you to easily
perform and detect post-translational
modiﬁcations by SUMO (small ubiquitinlike modiﬁer). With SUMOlink, you
simply add the assay components to a
microcentrifuge tube with your protein
of interest. After a 3-hour incubation,
the reaction is stopped and results canbe analyzed by Western blot (Figure 1).
With the kit’s p53 antibody and either
SUMO-1 or SUMO-2/3 antibodies, you
can easily see the extent to which yourtarget protein has been SUMOylated.

Everything you need to study SUMO
The kits contain E1 activating and E2
conjugating enzymes along with wildtype and mutant SUMO-1 (SUMO-1 Kit)
or SUMO-2 and -3 proteins (SUMO-2/3
Kit). Antibodies for SUMO-1 or SUMO2/3 modiﬁcations, as well as control p53
protein and antibody, are included. For
complete information, please give us acall or visit us at www.activemotif.com.

30
16
p53 antibody

SUMO-1 antibody

Figure 1: Enzymatic digestion of assembled chromatin.
Western blot analysis of in vitro SUMOylation of p53
protein by wild-type and mutated isoforms of SUMO-1:
The two Western blots were incubated with p53 antibody(1:5000 dilution) and SUMO-1 antibody (1 :4000 dilution).
p53 is SUMOylated only by wild-type SUMO-1 protein.
Lane 1: Wild-type SUMO protein conjugation reaction.
Lane 2: Mutated SUMO protein conjugation reaction.
Lane 3: No p53 control protein used in conjugation.

Product

FormatCatalog No.

SUMOlink™ SUMO-1 Kit

20 rxns

40120

SUMOlink™ SUMO-2/3 Kit

20 rxns

40220

Accurately Measure DNA Repair Protein Activity

The DNA Repair Protein Kit method
DNA Repair Protein Kits provide a quick
and easy method for sensitive DNA repair protein detection. Each kit contains
a 96-well plate with multiple copies ofan immobilized oligonucleotide containing a DNA lesion. Cell extract is added
to each well and the repair protein ofinterest binds speciﬁcally to the oligonucleotide on the plate. Each well isthen incubated with a primary antibody
directed against the repair protein being
studied. Addition of an HRP-conjugatedsecondary antibody and developing
solution provides an easily quantiﬁed
colorimetric readout (Figure 1).

www.activemotif.com

DNA Repair Protein Kit advantages• ELISA format eliminates gels,blotting and radioactivity• From cell extract to completed
assay in less than 5 hours• Up to 10-fold greater sensitivity
than gelshift assays• Ability to assay both cultured cells
and tissue samples
Product

A

GTBP
A

1.25
1
OD450 nmActive Motif’s DNA Repair Protein Kits are DNA-binding ELISAs that
detect DNA repair protein activity in both mammalian tissue and cell
culture extracts. They combine a fast, user-friendly format with a sensitive,
speciﬁc assay that is 10-fold more sensitive than gelshift, without the needfor gels or radioactivity. Quantitative results are achieved in less than 5 hours.

0.75
0.5
0.25

B

0

Raji

PANC-1

B

JurkatFigure 1: Measuring GTBP activity in several cell lines.
Jurkat, PANC-1 and Raji nuclear extracts were assessed
for GTBP activity using Western blot (A) and the GTBPDNA Repair Protein Kit (B). The DNA Repair Protein Kit isclearly more quantitative and sensitive.

FormatCatalog No.

GTBP DNA Repair Kit

1 x 96 well-plate5 x 96 well-plates

51096
51596

Ku70/86 DNA Repair Kit

1 x 96 well-plate5 x 96 well-plates

51196
51696

RPA DNA Repair Kit

1 x 96 well-plate5 x 96 well-plates

51296
51796

11

Epigenetics, Chromatin
Biology & Cell Signaling

THE NEWSLETTER OF ACTIVE MOTIF — March 2009 • volume 10 • number 1• Retroviruses, Chromatin Structure and Transcriptional Regulation• HeLa Acid Extracts for Epigenetic Studies

PRSRT STD
US POSTAGEPAID
PERMIT #94
SAN DIEGO, CA• Histones Containing Site- and Degree-speciﬁc Methylated Lysine Residues• Bisulﬁte Conversion for Accurate Analysis of Methylated DNA• Isolate CpG-Methylated DNA Using Recombinant Methyl-binding Protein• Upgrade your ChIP using Magnetic Bead-based ChIP-IT™ Express Kits
• Rigorous Testing Ensures Consistent Antibody Performance• Reconstitute Chromatin Quickly with the Chromatin Assembly Kit• Easily Purify Core Histones from Any Cell Culture or Tissue Sample• Assays to Measure HAT & HDAC Activity• In-cell ELISAs for More Quantitative Monitoring of Phosphorylation• Simpliﬁed Study of SUMOylation Effects with SUMOlink™
• Accurately Measure DNA Repair Protein Activity

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

Products for Epigenetics,
Chromatin Biology &Cell Signaling