THE NEWSLETTER OF ACTIVE MOTIF — November 2008 • volume 9 • number 3

Re-ChIP-IT™
your best bet
for sequential
immunoprecipitation(2 ChIPs)

2 Lower Background Co-IP of Both Nuclear & Whole-cell Complexes
3 NEW: Identify Protein Co-localization In Vivo Using Sequential Chromatin IP
4 NEW: Site- and Degree-speciﬁc Methylated Lysine Histone H3 & H4 Proteins

IN
THIS
ISSUE

5 NEW: Sensitive ELISAs to Quantitate Histone Methylation Levels
6 Rigorous Testing Ensures the Antibodies We Make Perform as Speciﬁed
7 NEW: Assays for Nrf2 and SREBP1 Activity
8 Rapid, Sensitive Assays for HAT & HDAC Activity and Inhibitor Compounds
9 NEW: Faster Histone Puriﬁcation with Convenient Spin Columns
10 Easily Compare CpG Methylation in Various DNA Samples
10 Reproducible Bisulﬁte Conversion for Accurate Analysis of Methylated DNA
11 Simple, Effective Monitoring of Phosphorylation Using In-cell Detection

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.

2

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 is 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 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

Toll Free — 1 877 222 9543

November 2008 • volume 9 • number 3

Sequential ChIP

NEW: Identify Protein Co-localization In Vivo Using Sequential Chromatin IP
Performing sequential chromatin IP (also called Re-ChIP) was technically 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 the same genomic locus.
Extend the utility of ChIP
When performing ChIP experiments, it isoften useful to prove that two different
proteins or histone modiﬁcations arepresent at the same site in the genome.
Or, you may want to determine if aprotein coincides with a speciﬁc histone
modiﬁcation at the same regulatory
element. Re-ChIP is a relatively newtechnique in which sequential chromatin
immunoprecipitations are performed
using two different antibodies, enabling
you to assay for the simultaneouspresence of two proteins or distinct
histone modiﬁcations at the same
genomic region of interest (Figure 1).
How does Re-ChIP-IT work?
The new Re-ChIP-IT Kit takes advantageof the same advances introduced in
Active Motif’s popular ChIP-IT Express
Kit. Each method uses protein G-coatedmagnetic beads that have less background than standard agarose beads, so
pre-clearing and blocking steps are notneeded. Magnetic pull-down occurs injust seconds, and the low backgroundhas eliminated the need for DNA puriﬁcation. With Re-ChIP-IT, the 1st ChIP is
performed as in regular ChIP. Chromatinthat has been immunoprecipitated isremoved from the magnetic beads witha special buffer that prevents the ﬁrst
antibody from participating in the 2nd
IP reaction. After desalting, a 2nd ChIP
is performed using an antibody different from that used in the ﬁrst ChIP.
The cross-links of these sequentiallyimmunoprecipitated protein-DNA complexes are then reversed and the DNA isanalyzed by PCR, similar to conventional
ChIP samples (Figure 2).

www.activemotif.com

Figure 1: Sequential chromatin immunoprecipitation using Re-ChIP-IT.
The lane numbers are the same in each panel to indicate that the DNA is from the same chromatin sample. The left panel
shows the results of PCR performed on an aliquot of DNA removed from the experiment after the first ChIP step; theright panel represents PCR results on DNA from chromatin samples after both ChIP steps. For example, chromatin samples
subjected to first ChIP using Mouse IgG as a negative control (lanes 1 and 2 in the left panel) were then subjected to a second ChIP with an RNA Pol II antibody (lanes 1 and 2 in the right panel). Chromatin samples in which Mouse IgG was used aseither the first antibody (lanes 1 and 2) or second antibody (lanes 5 and 6) show little amplification of GAPDH DNA in either
the left (first ChIP) or right panel (first and second ChIP). Chromatin samples in which the first antibody used was anti-RNA
Pol II and the second antibody was anti-TFIIB (lanes 3 and 4) show good amplification of GAPDH DNA after the second ChIP
(right panel) indicating co-localization of RNA Pol II and TFIIB at the same region of the GAPDH promoter.

Figure 2: Schematic representation of the Re-ChIP-IT procedure.

Product

FormatCatalog No.

Re-ChIP-IT™

25 rxns

53016

3

Methylated Histones

NEW: Site- and Degree-speciﬁc Methylated Lysine Histone H3 & H4 Proteins
Active Motif’s Recombinant Histones are available with site-speciﬁc mono-, di- and trimethylated lysines, so you
can investigate the inﬂuence of speciﬁc lysine methylation on the binding of associated chromatin proteins andnucleosome remodeling. In addition to the methylated histone H3 Lys4, 9 & 27 proteins released previously, we are
now introducing methylated histone H3 Lys79 & methylated histone H4 Lys20 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. To better
investigate how methylation patterns
impact regulatory processes, assays
will require histones with speciﬁcmethylation states. Active Motif recently
introduced methylated histone H3 (Lys4,
9 & 27) proteins, and we are now adding
methylated histone H4 Lys20 and H3
Lys79 proteins to our line-up. These
recombinant histones make it possible
to investigate the function of speciﬁcmethylation states on chromatin biology.

recombinant histone H4 proteins mono-,
di- and trimethylated at lysine 20.

binant histones are also analyzed by dot
blot or immunoblot to conﬁrm identity(Figure 2). The installed methylation statevery closely mimics natural methylation, so these recombinant histones are
perfect for any functional assay.

How is the methylation state made?
Using a patented approach, an analog ofmethyl lysine has been installed in thehistone via a chemical alkylation reaction. With this speciﬁc chemical treatment, the site and degree of methylation can be controlled. Each methylation
reaction is over 99% complete, andis veriﬁed by orthogonal acceleration
ESI-TOF mass spectrometry. The recomAnti-Histone H3 K9me1

Anti-Histone H3 K9me2

H3
H3
H3
K9me1 K9me2 K9me3

H3
H3
H3
K9me1 K9me2 K9me3

H3

Methylated histone H3 & histone H4
Active Motif is introducing recombinanthistone H3 proteins that are mono-,
di- and trimethylated at lysine 79 andOrder today!
Visit our website for more info or to try
any of these recombinant histones as astandard for your assays or as a building block for speciﬁc in vitro chromatinassays (Figure 1).

H3

Anti-Histone H3 K9me3
H3

36

36

36

30

30

30

16

16

16

H3
H3
H3
K9me1 K9me2 K9me3

6

6
6

1

2

3

4

5

6

7

8

9

10

11

12

Figure 2: Western blot analysis of mono-, di- and trimethylated Recombinant Histone H3.
1 µg Recombinant Histone H3 (Cat. No. 31207) (lanes 1, 5 & 9), 1 µg Recombinant Histone H3 monomethyl Lys 9 (Cat. No. 31211)(lanes 2, 6 & 10), 1 µg Recombinant Histone H3 dimethyl Lys9 (Cat. No. 31212) (lanes 3, 7 & 11) and 1 µg Recombinant Histone
H3 trimethyl Lys9 (Cat. No. 31213) probed with Histone H3 monomethyl Lys9 pAb (Cat. No. 39249) (lanes 1-4), Histone H3
dimethyl Lys9 pAb (Cat. No. 39239) (lanes 5-8) and Histone H3 trimethyl Lys9 pAb (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.

4

Product

FormatCatalog No.

Recombinant Histone H3 monomethyl Lys79

50 µg

31220

Recombinant Histone H3 dimethyl Lys79

50 µg

31221

Recombinant Histone H3 trimethyl Lys79

50 µg

31222

Recombinant Histone H4 monomethyl Lys20

50 µg

31224

Recombinant Histone H4 dimethyl Lys20

50 µg

31225

Recombinant Histone H4 trimethyl Lys20
50 µg
31226
Please visit www.activemotif.com/recomhistones to see our entire line of Recombinant Histones.

Toll Free — 1 877 222 9543

November 2008 • volume 9 • number 3

Histone Modification ELISAs

NEW: Sensitive ELISAs to Quantitate Histone Methylation Levels
Active Motif has applied our expertise in making histone-modiﬁcation antibodies to produce optimal antibody pairs
for the detection of speciﬁc histone modiﬁcations in a sandwich ELISA format. This makes it easy to quickly screenacid extracts for changes in levels of methylation of histone H3 at lysine 4. The kits include our site- and degreespeciﬁc methylated recombinant histones for use as a quantitative reference.
guaranteed (Figures 1 & 2). RecombinantHistone H3 di- or trimethyl Lys 4 protein(see Page 4) is included in the ELISA kits,
so you can compare sample results withknown protein amounts for quantitative
data on methylation levels.
What’s in the ELISA kits?
The Histone ELISA kits come completewith everything you need to quantify
changes in the levels of methylation ofhistone H3 at Lys4:• A 96 strip-well plate coated with
C-terminal histone H3 polyclonal
antibody for capture of histone H3• Biotinylated histone H3 di- ortrimethyl Lys4 antibody for speciﬁc
detection of di- or trimethylated H3• Recombinant Histone H3 di- ortrimethyl Lys4 protein for producing
standard curves• Streptavidin-HRP and all buffers &developing solutions required forcolorimetric readout

Histone ELISA advantages• Sensitive – works with 100-500 ng
of acid extract• Specific – C-terminal histone H3
capture and site- & degree-speciﬁcmethylation detection• Efficient – no time-consuming
immunoblotting• Convenient – strip-well format so
you use only what you needwww.activemotif.com

2.5

Histone H3
Histone H3 trimethyl Lys42

OD 450 (nm)Speciﬁc results fast!
Screening extracts by standard
immunoblotting methods is time consuming. Our new Histone Modiﬁcation
ELISAs enable you to efﬁciently screen
up to 96 wells at once, with little handson time, and to get colorimetric resultsin just hours. With Active Motif’s highquality modiﬁcation-speciﬁc histone
antibodies (see Page 6), accurate resultswith extremely low background are

Quantitate methylation levels
By including our unique RecombinantHistone H3 proteins with site- anddegree-speciﬁc methylation states,
methylation levels in sample acidextracts can easily be quantitated froma reference standard curve. Now you
can compare methylation state changes
in your samples without concerns over
speciﬁcity, and with less time and effort
than running immunoblots. Check our
website regularly for new releases ofRecombinant Histone H3 proteins andHistone Modiﬁcation ELISAs.

OD 450 (nm)Histone H3 Methyl Lys4
Methylation of Lys4 on histone H3
has been correlated with active genetranscription in eukaryotes, making theLys4 methylation state an interesting
marker of transcriptional activity. Active
Motif’s new Histone H3 methylated Lys4
ELISAs are now available for screening
the levels of di- or trimethyl Lys4 in acidextract samples. With the simple format
of our ELISA kit, it is easy to assess andquantitate levels of methylated histone
H3 Lys4 in acid extracts.

1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

Histone H3K4me1
Histone H3K4me2
Histone H3K4me3

80

40

20

10

5

2.5

1.25

Recombinant Histone H3 (ng)Figure 2: Histone H3 trimethyl Lys4 speciﬁcity.
1.25 to 80 ng of Recombinant Histone H3 mono-, di- andtrimethyl Lys4 proteins were assayed separately using
the Histone H3 trimethyl Lys4 ELISA kit. These results
indicate that the Histone H3 trimethyl Lys4 detection
antibody contained in Histone H3 trimethyl Lys4 ELISA
kit is highly specific, with little cross-reactivity formono- or dimethylated Lys4 on Histone H3. This meansthat small, specific changes in trimethyl Lys4 levels canbe detected with the ELISA.

1.5
1
0.5
0

125

62.5

31.25

15.62

7.81

3.90

1.95

0

Recombinant Histone H3 (ng)Figure 1: Histone H3 trimethyl Lys4 detection.
1.95 to 125 ng of Recombinant Histone H3 andRecombinant Histone H3 trimethyl Lys4 were assayedusing the Histone H3 trimethyl Lys4 ELISA.

Start screening modiﬁcation levels today!
Please visit us at www.activemotif.com
for complete information on our Histone
Modiﬁcation ELISAs, modiﬁcation-specific antibodies and additions to our line of
Recombinant Histone H3 proteins.

Product

FormatCatalog No.

Histone H3 dimethyl Lys4 ELISA

1 x 96 rxns

53102

Histone H3 trimethyl Lys4 ELISA

1 x 96 rxns

53103

5

Development of High-quality Antibodies

Rigorous Testing Ensures the Antibodies We Make Perform as Speciﬁed
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, manyof which have been validated for use in ChIP and immunoﬂuorescence (IF). Every antibody we make is tested rigorously
to ensure you are not wasting your precious time and research dollars on antibodies that don’t perform as speciﬁed. At
Active Motif, we believe that you should always repeat your experiments, but not because your reagents failed.
Our Antibody Commitment• Quality first – we’d rather fail our
project than sacriﬁce quality• Highly characterized– all of ourantibodies are tested stringently
under multiple conditions• Controlled process – we manufacture and test our own antibodies• Consistent – we go to great lengths
to minimize lot-to-lot variability

Figure 1: Dot blot analysis conﬁrms speciﬁcity.
Ten, 50 and 250 picomole aliquots of synthetic peptides
corresponding to histone H3 that is unmodified, mono-,
di- or trimethylated at Lys4 (Lanes 1-4), Lys9 (Lanes
5-8) and Lys27 (Lanes 9-12) were spotted and probedwith Histone H3 trimethyl Lys27 pAb (Cat. No. 39155),
confirming the antibody’s specificity for histone H3
trimethyl Lys27 (Lane 12). No cross-reactivity is observed
for the unmodified Lys27 peptide (Lane 9) and only ahint of signal is observed at the 250 picomole spots formono- (Lane 10) and dimethyl Lys27 (Lane 11).

Immunogen selection
Immunogens are selected to decrease
the likelihood of cross-reactivity withrelated proteins and to maximize detection of the protein in its native context.
Immunogens for modiﬁcation-speciﬁc
antibodies are selected to ensure thatthe antibody only recognizes the modiﬁed version of the protein.

6

Speciﬁcity screening
The ﬁrst test performed on every antibody is dot blot analysis, which ensures
its speciﬁcity for the desired protein or
modiﬁcation (Figure 1). Antibodies that
do not exhibit ≥ 25-fold selectivity forthe desired modiﬁcation are failed.
Western blot
Western blot is carried out to verify the
antibody recognizes the correct molecular weight protein and does not crossreact with other proteins. Commonlyused chemical treatments that stimulate
a modiﬁcation (e.g. HDAC inhibitors andacetylation) are used to better detect
the modiﬁcation (Figure 2).
Technique validation
Each antibody is validated for use intechniques such as ChIP (see Page 3) andIF (Figure 3) so that you have conﬁdence
when you use them in your experiments.

Figure 3: Immunoﬂuorescence testing.
HeLa cells stained with alpha Tubulin mAb (Clone 5-B1-2) in red (Cat. No. 39527, panels A and D), Histone H4monomethyl Lys20 pAb in green (Cat. No. 39175, B andD) and DAPI in blue (C and D). Note the strong nuclear
staining and absence of cytoplasmic staining in panel B,
as expected of an antibody to a histone modification.

Additional speciﬁcity testing
Antibodies to histone modiﬁcations thatexist in budding yeast (S. cerevisiae) canbe additionally screened for speciﬁcity
by testing strains of yeast that containa point mutation in the modiﬁed aminoacid (Figure 4).

Figure 4: Mutational analysis of yeast modiﬁcations.
Histone H4 acetyl Lys12 pAb (Cat. No. 39165) tested byWestern blot using extracts from wild-type yeast or
yeast containing a mutated histone H4 gene.

Figure 2: Western blot conﬁrming size & modiﬁcation.
Histone H2B acetyl Lys120 pAb (Cat. No. 39119) tested byWestern blot. No reactivity is observed on recombinanthuman histone H2B (Lane 1, 200 ng protein), as it is notacetylated. Lanes 2 and 3 contain 5 µg each of HeLa
cell acid extract. Cells that were treated with a histone
deacetylase inhibitor (sodium butyrate) prior to
extraction have an increased signal (Lane 3), as
expected for an antibody to a site of acetylation.

Lane 1: Yeast with wild-type histone H4.
Lane 2: H4 with the amino-terminal tail deleted.
Lane 3: H4 with an arginine instead of lysine 5.
Lane 4: H4 with an arginine instead of lysine 8.
Lane 5: H4 with an arginine instead of lysine 12.
Lane 6: H4 with an arginine instead of lysine 16.
Lane 7: H4 with arginines instead of lysines 5, 8 & 12.
Note the absence of reactivity in yeast extracts in which
lysine 12 is either missing or mutated (Lanes 2, 5 and 7).

Why not see for yourself?
For more information, use the convenient
search tools at www.activemotif.com/abs.

Toll Free — 1 877 222 9543

November 2008 • volume 9 • number 3

TransAM Transcription Factor ELISAs

NEW: Assay for Nrf2 and SREBP1 Activity

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.
1000

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
TransAM Kits have been cited in over
900 journal articles.
TransAM Nrf2
NF-E2 related factor (Nrf2) is a criticaltranscription factor in oxidative stresssignaling that may serve as a master
regulator in cellular defense pathways,
protecting a wide variety of tissues fromtoxic exposure. TransAM Nrf2 is a simple
solution that makes studying activationof Nrf2 in human, mouse and rat samples
more convenient (Figure 2).
1.6
1.4

Nrf2 activation (OD 450 nm)

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.

COS-7 unstimulated
COS-7 Nrf2 transfected

1.0
0.8
0.6
0.4

0

Citations

-0.2

600

1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
-0.2

COS-7 unstimulated
COS-7 SREBP1 transfected

0.6

1.25 2.55µg nuclear extracts

10

Figure 3: TransAM SREBP1 activation.
0.6 to 10 µg of COS-7 (SREBP1 transfected) nuclear
extract (red bars) and mock-transfected COS-7 nuclear
extracts (copper bars) were assayed per well.

TransAM SREBP1
TransAM SREBP1 (Sterol-regulatory element binding protein) detects SREBP-1aand -1c isoforms in as little as 0.6 µg
nuclear extract from human, mouse andrat samples (Figure 3).

1.2

0.2

800

SREBP1 activation (OD 450 nm)

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 Nrf2 and SREBP1.

0.15

0.3

0.63

1.25

2.5

µg nuclear extracts

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 us atwww.activemotif.com/transam for acomplete product listing, and to checkfor the latest releases!

Figure 2: TransAM Nrf2 activation.
0.15 to 2.5 µg of COS-7 (Nrf2 transfected) nuclear extract(red bars) and mock-transfected COS-7 nuclear extract(copper bars) were assayed per well.

400
200
0

TransAM™

Competitors

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.

www.activemotif.com

Product

FormatCatalog No.

TransAM™ Nrf2

1 x 96 rxns
5 x 96 rxns

50296
50796

TransAM™ SREBP1

1 x 96 rxns
5 x 96 rxn

50496
50996

7

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 free
Co-enzyme A generated by the histone
acetyltransferase activity to give a
ﬂuorescent readout. This makes it easy
to generate standard curves with acetylCoA or β-mercaptoethanol, so you canrelate the ﬂuorescence of your HAT topmol/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).

8

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 red 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

November 2008 • volume 9 • number 3

Histone Purification

NEW: Faster Histone Puriﬁcation With Convenient Spin Columns
Active Motif ﬁrst brought you the ground breaking Histone Puriﬁcation Kit, enabling fast and easy isolation ofcore histones from any cell culture or tissue sample. Now, we are expanding upon that innovative product byintroducing our new spin column-based Histone Puriﬁcation Mini Kit, which is even faster, more sensitive andeasier to use. Puriﬁcation of histone from yeast cells is also possible with this new kit. And, like the original kit,
the technique ensures that post-translational modiﬁcations remain intact.
How does it work?
Our unique method utilizes a puriﬁcation column with a proprietary binding matrix (Figure 1) and a series ofproprietary buffers to isolate very pure
fractions of histones. Using the Histone
Puriﬁcation Mini Kit, core histones canbe puriﬁed from cultured cells, yeast
cells and tissue samples as a single population containing H2A, H2B, H3 and H4(Figure 2). The column has a high afﬁnity
for histones, so histones can be puriﬁed from as little as 8x105 cells. While
you can not purify the same amountof histones as with the original Histone
Puriﬁcation Kit, spin columns make iteasier to isolate histones from multiple
samples simultaneously.

Figure 1: Histone puriﬁcation in a new spin column format.
Convenient spin columns enable faster purification ofhistones from multiple samples simultaneously.

Histone Puriﬁcation Mini advantages• Convenient spin column-based
protocol• Fast, efﬁcient, requiring less handson time• Prepare histones from multiple
samples simultaneously

www.activemotif.com

Figure 2: SDS-PAGE and Western blot analysis of histone fractions puriﬁed by the Histone Puriﬁcation Mini Kit.
Histones were purified from unstimulated, sodium butyrate-treated and paclitaxel-treated HeLa cells, rat brain tissue andbudding yeast. The 1st and 2nd elutions from each sample were analyzed on a 16% Tris-glycine SDS-PAGE gel (top panel). The
migration of the histones in the SDS gel is indicated by the bracket to the left of the gel. Westerns were then performedon the samples (bottom panel) using antibodies to acetyl-histone H3 (Ac-H3: Cat. No. 39139, 1:500 dilution), phospho-Ser28
histone H3 (Phos-H3: Cat. No. 39098, 1:1,000 dilution) and trimethyl-Lys4 histone H3 (Me-H3: Cat. No. 39159, 1:1,000 dilution).

Intact post-translational modiﬁcations
Preserving post-translational modiﬁcations like acetylation, methylation andphosphorylation is critical when investigating the role of histones in chromatin
biology. Our proprietary buffer systemis optimized to maintain these modiﬁcations while separating pure histone
fractions (Figure 2), which can be used as
substrates in many downstream assays,
analyzed by Western blot or mass spectrometry, or assembled into chromatinthat closely resembles native chromatin

for functional assays using Active Motif’s
Chromatin Assembly Kit (Cat. No. 53500).
Original kit now improved
Creating the spin-column format of the
new Histone Puriﬁcation Mini Kit has
inspired us to improve our original
Histone Puriﬁcation Kit, which can now
be used to purify all four core histones
using either the spin-column or gravityﬂow format, or as separate fractions of
H2A/H2B dimers and H3/H4 tetramers
using the gravity-ﬂow format.

Product

FormatCatalog No.

Histone Puriﬁcation Mini Kit

20 rxns

40026

Histone Puriﬁcation Kit

10 rxns

40025

9

DNA Methylation

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

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.

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).

10Advantages• 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

1

2

3

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

Toll Free — 1 877 222 9543

November 2008 • volume 9 • number 3

Phospho-Protein Detection

Simple, Effective Monitoring of Phosphorylation Using In-cell Detection

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™

www.activemotif.com

FACE™ Maker

FACE™ STAT4 (Y693)

11

Products for Chromatin &Transcriptional Regulation

THE NEWSLETTER OF ACTIVE MOTIF — November 2008 • volume 9 • number 3• Lower Background Co-IP of Both Nuclear & Whole-cell Complexes

PRSRT STD
US POSTAGEPAID
PERMIT #94
SAN DIEGO, CA• Identify Protein Co-localization In Vivo Using Sequential Chromatin IP• Site- and Degree-speciﬁc Methylated Lysine Histone H3 & H4 Proteins• Sensitive ELISAs to Quantitate Histone Methylation Levels• Rigorous Testing Ensures the Antibodies We Make Perform as Speciﬁed• Assays for Nrf2 and SREBP1 Activity• Rapid, Sensitive Assays for HAT & HDAC Activity and Inhibitor Compounds• Faster Histone Puriﬁcation with Convenient Spin Columns• Easily Compare CpG Methylation in Various DNA Samples• Reproducible Bisulﬁte Conversion for Analysis of Methylated DNA• Simple, Effective Monitoring of Phosphorylation Using In-cell Detection

Toll Free: 877 222 9543
Direct: 760 431 1263
Fax: 760 431 1351
Email: sales@activemotif.com
www.activemotif.com• Monitoring Phosphorylation Using In-cell Detection• Reproducible Bisulﬁte Conversion for Analysis of Methylated DNA• Easily Compare CpG Methylation in Various DNA Samples• Faster Histone Puriﬁcation with Convenient Spin Columns• Assays for HAT & HDAC Activity and Inhibitor Compounds• Assays for Nrf2 and SREBP1 Activity• Rigorous Testing Ensures Our Antibodies Perform as Speciﬁed• Sensitive ELISAs to Quantitate Histone Methylation Levels• Site- and Degree-speciﬁc Methylated Lysine Histone H3 & H4 Proteins• Identify Protein Co-localization In Vivo Using Sequential Chromatin IP• Lower Background Co-IP of Both Nuclear & Whole-cell Complexes

November 2008 • volume 9 • number 3

THE NEWSLETTER OF ACTIVE MOTIF

your best bet
for sequential
immunoprecipitation(2 ChIPs)

Re-ChIP-IT™

Products for Chromatin & Transcriptional Regulation