Products for

Chromatin Biology
andEpigenetics

Chromatin Immunoprecipitation (ChIP)
Histone Purification, DNA Methylation
Histone Antibodies, Histone ELISAs
Methylated Histone Proteins1

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Table of Contents
Epigenetics
Histone Acetylation and Genome Function
Review article by Dr. Jim Bone.......................................................3
Chromatin Modifier Antibodies
HDAC11......................................................................................................6
HMG-2......................................................................................................6
HP1 alpha..................................................................................................6
JARID1C.....................................................................................................6
JMJD2D.....................................................................................................6
JMJD2F.......................................................................................................6
L2MBTL1...................................................................................................6
LSD1............................................................................................................6
MBD1, -3, -4.............................................................................................6
MeCP2, -3................................................................................................6
MEP50.......................................................................................................6
Mi-2 beta.................................................................................................6
MRG15.......................................................................................................6
MTA2.........................................................................................................6
Nucleolin.................................................................................................6
SIRT1...........................................................................................................6
SNF2h........................................................................................................6
Suz12..........................................................................................................6
TRF2...........................................................................................................6
Histone & Histone Modification Antibodies
Histone H2A...........................................................................................7
Histone H2AX........................................................................................7
Histone H2A.Z.......................................................................................7
Histone H2B...........................................................................................7
Histone H3..............................................................................................7
Histone H4..............................................................................................7
Chromatin Immunoprecipitation
ChIP-IT™ Express...................................................................................8
ChIP-IT™ Express Enzymatic.............................................................8
ChIP-IT™ Control Kits..........................................................................8
ChIP-IT™ Protein G Magnetic Beads.............................................8
Ready-to-ChIP Chromatin................................................................8
ChIP-IT™ Express HT............................................................................9
Re-ChIP-IT™........................................................................................... 10
ChIP-IT™ Shearing Kit......................................................................... 11
Enzymatic Shearing Kit..................................................................... 11
Bridging Antibody for Mouse IgG............................................... 11

Histone ELISAsHistone H3 phospho Ser28 ELISA Kit........................................ 12
Histone H3 monomethyl Lys4 ELISA Kit.................................. 12
Histone H3 dimethyl Lys4 ELISA Kit.......................................... 12
Histone H3 trimethyl Lys4 ELISA Kit.......................................... 12
HeLa Acid Extracts............................................................................ 12
Chromatin Assembly
Chromatin Assembly Kit..................................................................13
Histone Purification
Histone Purification Kit................................................................... 14
Histone Purification Mini Kit (Spin Column Format)........... 15
Histone Acetylation & Deacetylation
HAT Assay Kit (Fluorescent)........................................................... 16
HDAC Assay Kits................................................................................. 16
Recombinant Methylated Histones
Histone H3............................................................................................ 17
Histone H3 mono-, di- & trimethyl............................................ 17
DNA Methylation
MethylDetector™............................................................................... 18
Fully Methylated Jurkat DNA........................................................ 18
MethylCollector™............................................................................... 19
Nuclear, Whole-cell & Cytoplasmic Extraction
Nuclear Extract Kit........................................................................... 20
Co-Immunoprecipitation
Universal Magnetic Co-IP Kit........................................................ 21
DNA Damage and Repair
DNA Damage Assay (Fluorescent).............................................. 22
Histone H2AX phospho Ser 139 pAb........................................ 22
Mitosis Determination
Mitotic Index Assay Kit...................................................................23
Mitotic Assay Kit (Color).................................................................23
Mitotic Assay Kit (Chemi)...............................................................23

Distributors
Distribution Network...................................................................... 24

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c h r o m at i n b i o l o g yHistone Acetylation and Genome Function: Transcription and BeyondThe role of histone acetylation and its involvement in
the regulation of transcription has long been a topic
of research in cell and molecular biology labs. Recent studies have revealed the role of histone acetylationin other important processes regulating the structure andfunction of chromatin, and hence, the eukaryotic genome.
The process of organizing the millions (billions in thecase of humans) of base pairs of genetic material in the
eukaryotic nucleus has profound effects on DNA-dependent events, such as transcription, recombination, replication and repair. DNA is organized by its incorporation
into chromatin, the basic subunit of chromatin being thenucleosome. A nucleosome is composed of 147 base pairsof DNA coiled around an octamer of histone proteins, twomolecules each of histone H2A, H2B, H3 and H4. Histone
H1 associates with chromatin outside the core octamer
unit and regulates higher order chromatin structure. Chromatin and chromosomes undergo dramatic and dynamicchanges in organization in response to a myriad of cellularsignals. Chromosomes condense and relax during the cell
division process. Damaged DNA adopts a unique structurethat facilitates repair. Critical to cell function, most of the
genome must remain in a transcriptionally silent state, savefor specific combinations of genes, which vary significantly between cell types. These processes must be tightly
regulated to maintain the integrity of the genome and theproper function of the cell.
Multiple mechanisms exist within the nucleus thatallow the function and organization of the genome to be
dynamically regulated, responding rapidly to different signals or inputs. Most notably, though, a large body of evidence dating back forty years and more has accumulated
to indicate that post-translational modification of histones
is crucial to all genome-based activity (see Kouzarides,2007). The recruitment of transcriptional regulators andchromosomal proteins brings along enzymes that modify(by either addition or removal) specific functional groups
on histones, and these dynamic addition and subtraction
events have profound effects on the structure and function of chromatin.
Histone Acetylation and Transcription
The most widely studied histone modification is acetylation. Histones are covalently modified at the epsilon-amino

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Jim Bone, Ph.D.

group of conserved lysines by a class of enzymes called
histone acetyltransferases (HATs). HATs come in two flavors,
cytoplasmic and nuclear. The cytoplasmic HATs (e.g. Hat1)
acetylate histones prior to nuclear localization and chromatin assembly, whereas the nuclear HATs acetylate histones
in a manner associated with transcription and other DNAdependent processes.
Some of the earliest observations made by Allfrey
and colleagues linked histone acetylation to transcription(Allfrey et al., 1964). A large amount of descriptive and correlational evidence accumulated in the thirty years after
this initial work, until the identification of the first nuclear
histone acetyltransferase, Tetrahymena p55, provided thefirst mechanistic link between histone acetylation andtranscriptional activation (Brownell et al., 1996). The yeast
homologue of p55, Gcn5, had previously been identified
as an important transcriptional co-activator, a protein
recruited to gene promoters prior to transcription andnecessary for activation. Further solidifying the connection between histone acetylation and transcription, it wasdetermined that the HAT activity of Gcn5 is required for
its ability to activate transcription (Candau et al., 1997; Kuo
et al., 1998).

Tetrahymena p55 provided thefirst mechanistic link between
histone acetylation andtranscriptional activation.
Biochemical analysis of a variety of proteins and protein
complexes involved in transcriptional activation led to theidentification of a large number of histone acetyltransferases, strengthening the hypothesis that histone acetylation istightly connected to (and likely part and parcel of) transcriptional activation (reviewed in Roth and Denu, 2001). Additional support for this hypothesis came from the opposite
side of the histone acetylation coin, histone deacetylation.
Contemporaneous with the identification of Gcn5 as thefirst HAT, the mammalian homologue of a known transcriptional repressor from yeast, Rpd3, was identified as the first
enzyme that removes epsilon-acetylation from histones, a

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Histone Acetylation and Genome Function, cont.
histone deacetylase, or HDAC (Taunton et al., 1996). Runningparallel to the advances made with HATs, a wide variety of
HDACs and HDAC-containing protein complexes were identified and linked to the repression of transcription (reviewed
Yang and Seto, 2003).

nant chromatin acetylated homogeneously, it was determined that acetylation of lysine 16 inhibits the formationof higher-order chromatin. This result suggests that theunique role played by lysine 16 is to serve as a central regulator of chromatin structure (Shogren-Knaak et al., 2006).

Transcriptional Elongation
While the connection between histone acetylation andtranscriptional activation has been known for some time, it
has only recently been established that histone acetylation
is involved in the process of transcriptional elongation.
Whereas promoters of active genes are highly acetylated,the coding regions of genes need to be maintained in a
hypoacetylated state to prevent the aberrant initiationof transcription. A histone methyltransferase (Set2) thattravels with the elongating RNA polymerase marks thecoding sequence with a unique modification, histone H3
methylated at lysine 36. A histone deacetylase complex(Rpd3S) is recruited and specifically deacetylates coding region chromatin through the recognition of lysine 36 by thechromodomain protein, Eaf3, a subunit of Rpd3S (Carrozzaet al., 2005; reviewed in Lee and Shilatifard, 2007).

Genome Integrity, DNA Replication & DNA Damage Repair
There is a recent and growing body of evidence linking
histone acetylation to DNA replication and repair. Oneof the first studies suggesting a link between acetylation
and DNA repair came from a genetic study of histone H4acetylation in yeast. A yeast strain containing mutations in
the four amino-terminal lysines of histone H4 has severelycompromised genome integrity, leading to continual
activation of the DNA damage repair checkpoint (Megee
et al., 1995). A more recent study showed that this same
yeast strain exhibits a dramatically reduced ability to repair
double-strand breaks. It was determined that the histone
acetyltransferase complex NuA4 was recruited to the siteof double-strand breaks. Efficient repair of DNA damage
was shown to require the catalytic subunit of the NuA4,
Esa1, a HAT enzyme that acetylates histone H4. Thus, notonly are proper levels of histone H4 acetylation required
for maintaining genome integrity, it appears that acetylation of histone H4 participates directly in one or more
steps required to repair broken chromatin (Bird et al., 2002).

Lys 16: An Acetylation Site with a Unique Molecular Identity
With regard to transcriptional activation, the acetylation of specific lysines appears to be less important thanthe overall acetylation level of histone proteins on thewhole. There appears to be a functional redundancy
between lysine residues, with no single lysine being moreor less critical than the next. Indeed, HAT enzymes exhibit broad substrate specificity, although are apparently
limited by which histone they are capable of acetylating.
The exception to this rule is lysine 16 of histone H4, theacetylation and deacetylation of which have specific roles
in important transcriptional processes (reviewed in Shia etal., 2006). In yeast, acetylation of histone H3 at lysine 16 isrequired to maintain the boundaries between euchromatin(open and transcriptionally competent regions of chromatin) and heterochromatin (closed and transcriptionallyinert). In Drosophila, lysine 16 acetylation is involved in theprocess of dosage compensation, the coordinate upregulation of genes on the single male X chromosome that
equalizes gene expression with that of genes on the two
X chromosomes in females. In humans, the loss of lysine
16 acetylation appears to be a hallmark of specific typesof cancer. The common thread between these findings
may be found in the results of a recent study employing aunique biochemical method to study histone acetylation.
Using a peptide-ligation method to synthesize recombi-

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FIGURE 1:
Immunofluorescence of HeLa cells to visualize histone H3 acetylated at Lys 27.
HeLa cells were grown, fixed and stained using the MAX Stain™ Universal Immunofluorescence System (Catalog No. 15250). The primary antibody used was theHistone H3 acetyl Lys27, symmetric pAb (Catalog No. 39133) at a dilution of 1:1,000.

The involvement of histone acetylation in DNA damage repair extends to mammals as well. The mammalianEsa1 homologue, TIP60, is involved with many aspects of
the DNA damage repair pathway (reviewed in Squatrito etJAPAN +81 (0)3 5225 3638

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c h r o m at i n b i o l o g yal., 2006). In addition to a direct role in the repair of DNA
damage similar to that of Esa1 (Murr et al., 2006), TIP60 isalso involved in the activation of the DNA damage repair
pathway through the acetylation and activation of the
ATM kinase (Sun et al., 2005). TIP60 also acetylates the
DNA damage-specific histone variant, H2AX, in response
to DNA damage. Acetylation of H2AX leads to its subsequent ubiquitylation and remodeling of chromatin near
the break, facilitating DNA repair (Ikura et al., 2007).
The fascinating connection between histone acetylation and DNA damage repair has been made even moretantalizing by the identification of the importance of asingle acetylation site in the process. This same single
acetylation event is also required for DNA replication andchromatin assembly, which are associated with replicationcoupled DNA repair. Li and colleagues found that acetylation of lysine 56 in yeast plays a specific role in chromatin
assembly, dependant upon chromatin assembly factor-1(CAF-1), a histone chaperone complex (Li et al., 2008). It
was determined that acetylation of lysine 56 increases
the affinity of CAF-1 for histone H3, thus improving theefficiency of chromatin assembly. In a related study, it
was found that acetylation of lysine 56 (and downstream
chromatin assembly) is linked to proper cell cycle re-entry
subsequent to double-strand DNA break repair (Chen etal., 2008). The histone chaperone Asf1 (acting at a point
before CAF-1) stimulates the acetylation of histone H3 atlysine 56 prior to its transfer to the nucleus and incorporation into chromatin. Lysine 56 acetylation is required forthe timely deactivation of the DNA damage checkpoint
after DNA repair is complete, allowing cells to re-enter thecell cycle. This suggests that the presence of H3 acetylated
at lysine 56 is a hallmark of the properly re-assembled
chromatin at the site of a newly repaired double-strand
break, allowing the inactivation of the DNA damage
checkpoint. However, in separate studies it was determined that unregulated lysine 56 acetylation is deleterious to cells and leads to spontaneous DNA damage (Celic
et al., 2008; Haldar and Kamakaka, 2008). It is possiblethat this is also the case in mammals as well as in yeast,as hyperacetylation of histones leads to the activation of
the ATM DNA damage checkpoint protein (Bakkenist andKastan, 2003). In addition to the specific role of acetylation
at lysine 56 of histone H3 to facilitate chromatin assembly and serve as a marker for newly repaired chromatin,
global acetylation levels must be kept at an intermediate,highly regulated level in order to maintain proper genome
structure.

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Involved as it is in the mechanism of DNA replicationand chromatin assembly, there is evidence that histone
acetylation also plays a role in the timing of DNA replication and replication origin activity. In general, increases inhistone acetylation of chromatin surrounding an origin of
replication tend to cause the origin to initiate replication
earlier, compared to when the origin is within hypoacetylated chromatin (reviewed in Weinreich et al., 2004). Origins located near or within heterochromatin replicate late,but this timing can be accelerated by artificially increasing
the acetylation state of histones near the origin. As origin
firing occurs throughout S-phase, rather than clustered
at one time point, it is likely that individual origin firing is
influenced by the local chromatin environment surrounding the origin (Vogelauer et al., 2002).
Future Directions
This review has discussed many of the important rolesof histone acetylation with regard to regulating genome
function, however many more questions have yet to beanswered. With regard to etiology, what is the relationship between acetylation and transcriptional activation?
Are there other acetylation sites with unique molecular
identities, such as H3 lysine 56 and H4 lysine 16? And
importantly, what are the molecular mechanisms by whichacetylation mediates its diverse functions? Acetylation isthe best studied of the histone modifications, but there ismuch more remaining to be learned.
REFERENCES
Allfrey, V.G. et al. (1964) PNAS 51: 786-794.
Bakkenist, C.J. and Kastan, M. (2003) Nature 421: 499-506.
Bird, A.W. et al. (2002) Nature 419: 411-415.
Candau, R. et al. (1997) EMBO J 16: 555-565.
Carrozza, M.J. et al. (2005) Cell 123: 581-592.
Celic, I. et al. (2008) Genetics 179: 1769-1784.
Chen, C.C. et al. (2008) Cell 134: 231-243.
Haldar, D. and Kamakaka, R. (2008) Euk Cell 7: 800-813.
Ikura, T. et al. (2007) Mol Cell Biol 27: 7028-7040.
Kouzarides, T. (2007) Cell 128: 693-705.
Kuo, M.H. et al. (1998) Genes Dev 12: 627-639.
Lee, J.S. and Shilatifard, A. (2007) Mutat Res 618: 130-134.
Li, Q. et al. (2008) Cell 134: 244-255.
Megee, P.C. et al. (1995) Genes Dev 9: 1716-1727.
Mottet, D. and Castronovo, V. (2008) Clin Exp Metastasis 25: 183-189.
Murr, R. et al. (2006) Nature Cell Biol 8: 91- 99.
Roth, S.Y. et al. (2001) Annu Rev Biochem 70: 81-120.
Shia, W.J. et al. (2006) Genome Biol 7: 2507-2512.
Shogren-Knaak, et al. (2006) Science 311: 844-847.
Squatrito, M. et al. (2006) Trends Cell Biol 16: 433-442.
Sun, Y. et al. (2005) PNAS 102: 13182-13187.
Taunton, J. et al. (1996) Science 272: 408-411.
Vogelauer, M. et al. (2002) Mol Cell 10: 1223-1233.
Weinreich, M. et al. (2004) Biochim Biophys Acta 1677: 142-157.
Yang, X.J. and Seto, E. (2003) Curr Opin Genet Dev 13: 143-153.

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Antibodies
Antibodies to Study Chromatin and the Biology of the Nucleus
high-quality antibodies to histones, histone modifications and chromatin-modifying proteins
At Active Motif, we are committed to providing researcherswith the highest quality antibodies for studying chromatin andthe biology of the nucleus. We manufacture our to histone
and histone modification antibodies in-house, so we control

antibody quality and performance. We also offer a wide range ofantibodies against transcription factors, chromatin modifiers andcell-cycle regulators.

The Active Motif antibody difference• Quality first – we’d rather fail our antibody project
than sacrifice quality• Highly characterized – all antibodies aretested stringently under multiple conditions• Controlled process – we manufacture andtest all our own antibodies• Consistent – we go to great lengths to minimize
lot-to-lot variability

FIGURE 1:
Chromatin immunoprecipitation.
ChIP was performed with Histone H3 dimethyl Lys4 pAb (Catalog No. 39141) using
chromatin prepared from mouse embryonic stem cells (left lane) or mouse erythroleukemia cells (right lane). PCR primers specific for the b-globin gene were used
to amplify a 210 base pair region of the promoter following DNA isolation.

• Convenient – most antibodies are available intwo pack sizes

Antibodies to Chromatin Modifiers
There are many important non-histone chromatin proteins, from
histone modifying enzymes like acetyltransferases, deacetylases
and methyltransferases to chromatin binding proteins like HP1,
which reads the “histone code”.

Because these proteins are important regulators of chromatin
structure and function, Active Motif offers high-quality
antibodies to these targets so you can study their roles.

DESCRIPTIONAPPLICATIONS CAT. NO.

DESCRIPTIONAPPLICATIONS CAT. NO.

HDAC11 Rabbit pAb
HMG-2 Rabbit pAb
HP1 alpha Rabbit pAb NEW
JARID1C Rabbit pAb NEW
JMJD2D Rabbit pAb NEW
JMJD2F Rabbit pAb NEW
L3MBTL1 Rabbit pAb
LSD1 Rabbit pAb
MBD1 Mouse mAb
MBD3 Mouse mAb
MBD4 Rabbit pAb

WB
WB
ChIP, IP, WB
WB
WB
WB
ChIP, IP, WB
IP, WBWB
WBWB

MeCP2 Rabbit pAb
MeCP3 Rabbit pAb
MEP50 Rabbit pAb
Mi-2 beta Rabbit pAb NEW
MRG15 Rabbit pAb NEW
MTA2 Rabbit pAb NEW
Nucleolin Mouse mAb (Clone 3G4B20)
SIRT1 Mouse mAb (Clone 2G1/F7) NEW
SNF2h Mouse mAb
Suz12 Rabbit pAb NEW
TRF2 Goat pAb

WB
WB
WB
ChIP, IF, IP, WB
WB
WB
IF, WBIF, IP, WB
ChIP, IF, IP, WB
WB
ChIP, IP, WB

39208
39029
39295
39229
39247
39257
39182
39186
39215
39216
39217

39188
39218
39190
39289
39361
39359
39541
39353
39543
39357
39223

APPLICATIONS KEY: ChIP = Chromatin Immunoprecipitation; IF = Immunofluorescence; IP = Immunoprecipitation; WB = Western Blot

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Antibodies

c h r o m at i n b i o l o g yHistones and Histone Modification Antibodies

ChIP-validated Antibodies

Histone post-translational modifications are important regulatorsof genome function. Active Motif is working to develop a panelof antibodies for all widely studied and biologically relevant
modifications sites. All of our antibodies are rigorously screened
for specificity and tested to verify which applications they work
well in, such as ChIP, Western blotting and immunofluorescence.

Chromatin immunoprecipitation is an extremely challenging
technique, and only antibodies of the highest quality will do. For
an antibody to work in chromatin IP, it needs to be of high titer,and be very specific, with no detection of non-target proteins.
The most important characteristic is that it also has to recognize
the target protein in its native context.

DESCRIPTIONDESCRIPTIONAPPLICATIONS CAT. NO.

Histone H2A Rabbit pAb
WB
Histone H2A Rabbit pAb (yeast)
ChIP, WB
Histone H2A, acidic patch Rabbit pAb
WB
Histone H2A acetyl Lys5 Rabbit pAb
WB
Histone H2A acetyl Lys9 Rabbit pAb
WB
Histone H2A phospho Ser129 Rabbit pAb (yeast) NEW ChIP, IF, IP, WB
Histone H2A/H4 phospho Ser1 Rabbit pAb
WB
Histone H2AX phospho Ser139 Rabbit pAb
IF, WB
Histone H2A.Z Rabbit pAb
WB
Histone H2B Rabbit pAb
WB
Histone H2B Rabbit pAb
WB
Histone H2B Rabbit pAb (yeast)
ChIP, WB
Histone H2B acetyl Lys5 Rabbit pAb
WB
Histone H2B acetyl Lys16 Rabbit pAb
WB
Histone H2B acetyl Lys120 Rabbit pAb
WB
Histone H3 Rabbit pAb
WB
Histone H3, C-terminal Rabbit pAb
ChIP, WB
Histone H3 acetyl Rabbit pAb
WB
Histone H3 phospho Thr3 Rabbit pAb
WB
Histone H3 monomethyl Lys4 Rabbit pAb NEW
WB
Histone H3 dimethyl Lys4 Rabbit pAb
ChIP, WB
Histone H3 trimethyl Lys4 Rabbit pAb
ChIP, WB
Histone H3 acetyl Lys9 Rabbit pAb
WB
Histone H3 pan-methyl Lys9 Rabbit pAb NEW
IF, WB
Histone H3 monomethyl Lys9 Rabbit pAb NEW
IF, WB
Histone H3 dimethyl Lys9 Mouse mAb (Clone 6F12-H4) ChIP, IF, IP, WB
Histone H3 dimethyl Lys9 Rabbit pAb
ChIP, IF, WB
Histone H3 trimethyl Lys9 Rabbit pAb
WB
Histone H3 phospho Ser10 Rabbit pAb NEW
IF, WB
Histone H3 phospho Ser10,28 Rabbit pAb
WB
Histone H3 phospho Thr11 Rabbit pAb
WB
Histone H3 acetyl Lys14 Rabbit pAb
WB
Histone H3 dimethyl Lys14 Rabbit pAb NEW
WB

39209
39235
39111
39107
39109
39271
39115
39117
39113
39125
39210
39237
39123
39121
39119
39088
39163
39139
39153
39297
39141
39159
39137
39241
39249
39285
39239
39161
39253
39147
39151
39127
39349

APPLICATIONS CAT. NO.

Histone H3 acetyl Lys18 Rabbit pAb
IF, WB
Histone H3 acetyl Lys23 Rabbit pAb
IF, WB
Histone H3 acetyl Lys27 Rabbit pAb
ChIP, WB
Histone H3 acetyl Lys27 Rabbit pAb
ChIP, IF, WB
Histone H3 dimethyl Lys27 Rabbit pAb NEW
IF, WB
Histone H3 trimethyl Lys27 Mouse mAb
IF, WB
Histone H3 trimethyl Lys27 Mouse mAb
ChIP, WB
Histone H3 trimethyl Lys27 Rabbit pAb
IF, WB
Histone H3 trimethyl Lys27 Rabbit pAb
WB
Histone H3 phospho Ser28 Mouse pAb
WB
Histone H3 phospho Ser28 Rabbit pAb
WB
Histone H3 phospho Ser28 Rat mAb (Clone HTA28)
IF, WB
Histone H3 dimethyl Lys36 Rabbit pAb NEW
IF, WB
Histone H3 acetyl Lys56 Rabbit pAb NEW
WB
Histone H3 monomethyl Lys56 Rabbit pAb NEW
WB
Histone H3 dimethyl Lys56 Rabbit pAb NEW
WB
Histone H3 monomethyl Lys79 Rabbit pAb
WB
Histone H3 monomethyl Lys79 Rabbit pAb NEW
WB
Histone H3 dimethyl Lys79 Rabbit pAb
WB
Histone H4 Rabbit pAb
WB
Histone H4 pan-acetyl Rabbit pAb NEW
IF, WB
Histone H4 tetra-acetyl Rabbit pAb
IF, WB
Histone H4 tetra-acetyl Rabbit pAb
ChIP, WB
Histone H4 dimethyl Arg3, symmetric Rabbit pAb NEW IF, WB
Histone H4 acetyl Lys5 Rabbit pAb
ChIP, IF, WB
Histone H4 acetyl Lys8 Rabbit pAb
WB
Histone H4 acetyl Lys12 Rabbit pAb
ChIP, WB
Histone H4 acetyl Lys16 Rabbit pAb
ChIP, WB
Histone H4 monomethyl Lys20 Rabbit pAb
ChIP, IF, WB
Histone H4 dimethyl Lys20 Rabbit pAb
IF, WB
Histone H4 dimethyl Lys20 Mouse mAb
WB
Histone H4 trimethyl Lys20 Rabbit pAb
ChIP, IF, WB

39129
39131
39135
39133
39245
39536
39535
39155
39156
39211
39149
39098
39255
39281
39273
39277
39145
39367
39143
39212
39243
39177
39179
39275
39169
39171
39165
39167
39175
39173
39539
39180

APPLICATIONS KEY: ChIP = Chromatin Immunoprecipitation; IF = Immunofluorescence; IP = Immunoprecipitation; WB = Western Blot

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Chromatin Immunoprecipitation
ChIP-IT™ Express
improved kits greatly reduce background
Active Motif has improved its ChIP-IT™ Express Kits by greatly
reducing the background, which improves your results andenables you to use less starting material than ever before. In
addition, the provided magnetic beads have made it possible toChIP-IT Express advantages

streamline the protocol so you can get results in half the normal
time with much less sample manipulation. Also, ChIP-IT Express
makes it easy to perform ChIP on many samples simultaneously.

The magnetic bead advantage• No more need for pre-clearing, blocking or DNA
purification steps

The ChIP-IT Express magnetic beads have less background thanstandard agarose beads, so pre-clearing and blocking steps are nolonger necessary. The magnetic pull-down occurs in just seconds,
and re-formulated buffers allow steps to be combined and DNA
purification to be eliminated. ChIP-IT Express is available in bothsonication and enzymatic shearing formats.

• Reduced background• High throughput compatible• Dramatically reduced hands-on time

The most efficient ChIP enrichment kit

Positive controls ensure success

ChIP is an enrichment technique, not a purification method.
Thus, the less efficient your enrichment, the higher the sample
background and the more material you will need to obtain aninterpretable result. Conventional ChIP requires at least 2 million cells as starting material, which can be problematic withsome cell lines. At the least, growing this many cells is labor intensive. Active Motif’s improved ChIP-IT Express Kits, however,
have been optimized to provide superior target gene enrichment, resulting in unmatched sensitivity. Using ChIP-IT Express,
it is routine to perform ChIP on material from as few as 750,000
cells and the kits have even been shown to work with as few as
12,500 cells (Figure 1).

Because interpreting ChIP data can be difficult, Active Motif
has developed a complete set of controls to help you understand your results and troubleshoot your assays. To provide
you with controls that are appropriate for your research, weoffer our human, mouse and rat ChIP-IT Control Kits separatelyfrom ChIP-IT Express Kits. These provide positive and negative
control antibodies and species-specific primers, PCR buffer anda convenient 10X DNA loading dye so your PCR reactions are
gel-ready. All reagents are quality control tested and validated toensure your ChIP assay is working properly. In addition, we alsooffer convenient Ready-to-ChIP Chromatin from a number of
ENCODE cell lines, so you can be certain that the only variable invalidating a new antibody for ChIP is the antibody itself.

25,000 cells

Neg IgG

RNA pol II

Neg IgG

RNA pol II

Neg IgG

RNA pol II

Neg IgG

RNA pol II

GAPDH Primers

12,500 cells

FIGURE 1:
ChIP-IT Express works with 12,500 cells.
ChIP-IT Express was performed in duplicate on decreasing amounts of sonicated
HeLa cell chromatin. Two µg of RNA pol II and Neg IgG antibody was used for IP.
GAPDH PCR primers were used to analyze the immunoprecipitated DNA. Using
the improved ChIP-IT Express reagents and protocol, positive ChIP data was
obtained from as few as 12,500 cells.

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Try the best ChIP kit today
For additional information on the new and improved ChIP-IT
Express Kits, visit our website at www.activemotif.com/chipit.
Product

FormatCat. No.

ChIP-IT™ Express

25 rxns

53008

ChIP-IT™ Express Enzymatic

25 rxns

53009

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

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ChIP

c h r o m at i n b i o l o g y

ChIP-IT™ Express HT
process up to 96 ChIP reactions at a time
Active Motif now makes it easy for you to perform your ChIP
experiments on a high-throughput scale with the introductionof ChIP-IT™ Express HT. Based on the innovative ChIP-IT Express

Kit, it uses the time-saving magnetic bead method for faster andcleaner chromatin IP experiments, enabling you to process up to
96 ChIP reactions at once.
FIGURE 1:
Chromatin IP performed on HeLa
chromatin with ChIP-IT Express HT.
PCR carried out using primers
specific for the GAPDH gene.
Lanes 1-4: ChIP using 2 µg RNA
Pol II antibody. Lanes 5-8: ChIP
using normal mouse IgG as anegative control. Lane 9: noDNA control. Lane 10: input DNA
control.

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 thetime-saving, magnetic bead-based protocol of ChIP-IT Expresswith a high-throughput, 96-well microplate-based 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 as with the ChIP-IT Control Kits.

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.

Improved ChIP
Chromatin Immunoprecipitation (ChIP) is an important technique, enabling the identification of the in vivo sites of chromatin binding proteins, or localization of histone modifications to
specific regions of the genome. Recent improvements in ChIP
protocols and reagents have made ChIP experiments faster andeasier to perform, as well as more successful. In addition, thelower background enabled by improved ChIP techniques has
made it 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 ofmagnetic beads for ChIP in its ChIP-IT Express Kit. The ChIP-IT
Express HT Kit is the next stage in the development of kits forchromatin IP.

For additional information on the
new ChIP-IT Express HT Kit, go to:www.activemotif.com/htchip.

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FREE 96-wellmagnetic sorter!
Order ChIP-IT Express HTand receive a free 96-wellmagnetic sorter. Available
while supplies last.

Product

FormatCat. No.

ChIP-IT™ Express HT

96 rxns

53018

EUROPE +32 (0)2 653 0001

NORTH AMERICA 877 222 9543

9

Chromatin Immunoprecipitation
Re-ChIP-IT™
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
Re-ChIP-IT™ Kit makes it easy to perform sequential ChIP, so you

can localize two different proteins or histone modifications tothe same genomic locus.

Extend the utility of ChIP
When performing chromatin immunoprecipitation (ChIP) experiments, it is often useful to prove that two different proteinsor histone modifications are present at the same site in the
genome. Or, you may want to determine if a protein coincides
with a specific histone modification at the same regulatory
element. Re-ChIP (aka Sequential ChIP, Chromatin Re-IP and
ChIP Re-ChIP) is a relatively new technique that enables
sequential chromatin immunoprecipitations to be performed
using two different antibodies, enabling you to you assay forthe simultaneous presence of two proteins or distinct histone
modifications at the same genomic region of interest.
Active Motif’s new Re-ChIP-IT Kit makes it simple for you to
perform these types of experiments. All buffers for chromatin
IP are included, making it easy to get started. And, the detailed
protocol ensures you’re successful the first and every time. Plus,
Active Motif offers a variety of chromatin IP control kits to help
you validate the results of your Re-ChIP experiments.

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

Sequential chromatin IP made easy
Re-ChIP-IT uses magnetic beads that have less background thanstandard agarose beads, so pre-clearing and blocking steps are
not needed. Magnetic pull-down occurs in just seconds, and themethod’s low background has eliminated the need for DNA
purification. And, Re-ChIP-IT can be used with chromatin
prepared using our sonication or enzymatic shearing kits.

FIGURE 1:
Sequential chromatin immunoprecipitation using Re-ChIP-IT.
The lane numbers are the same in each panel to indicate that the DNA is fromthe same chromatin sample. The left panel shows the results of PCR performedon an aliquot of DNA removed from the experiment after the first ChIP step;
the right 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 thensubjected to a second ChIP with an RNA Pol II antibody (lanes 1 and 2 in theright panel). Chromatin samples in which Mouse IgG was used as either the firstantibody (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 secondChIP). 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 amplificationof 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.

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Get started with Re-ChIP
For additional information on the new Re-ChIP-IT Kit go towww.activemotif.com/rechip.

Product

FormatCat. No.

Re-ChIP-IT™

25 rxns

53016

JAPAN +81 (0)3 5225 3638

www.activemotif.com

ChIP

c h r o m at i n b i o l o g y

ChIP Accessory Kits and Reagents
maximize your chromatin IP experiments
ChIP & IP enhancer for mouse antibodies

Beads, controls and shearing kits

Low antibody binding affinity can make getting good ChIP & IP
results difficult. Because mouse IgM and IgG1 antibodies do nothave strong binding affinity for protein G, ChIP and regularimmunoprecipitation (IP) can be challenging when using protein
G-conjugated beads. The Bridging Antibody for Mouse IgG
facilitates improved binding of mouse antibodies, especially IgG1
and IgM isotypes, to protein G to generate more robust binding
and better results in ChIP and standard IP experiments (Figure 1).

Active Motif offers a broad range of reagents and accessory kits
to complement the ChIP-IT™ line of ChIP kits. These products
will help you prepare chromatin, troubleshoot your ChIP experiments and make them more reproducible.

1

2

3

4

5

6

7

FIGURE 2:
Analysis of DNA sheared using the
Enzymatic Shearing Kit.
HeLa cells were fixed for 10 minuteswith 1% formaldehyde and thenchromatin was prepared using the
Enzymatic Shearing Kit protocol.
Chromatin was sheared with the
Enzymatic Shearing Cocktail for 5, 10& 15 minutes and the reaction was
stopped.

8

Lane 1: 100 to 1000 bp ladder.
Lane 2: Unsheared HeLa DNA.
Lane 3: HeLa DNA treated for
5 minutes.
Lane 4: HeLa DNA treated for
10 minutes (optimized).
Lane 5: HeLa DNA treated for
15 minutes.

Ready-to-ChIP Chromatin
FIGURE 1:
Improvement in chromatin IP using an anti-mouse bridging antibody.
ChIP was performed using chromatin from U-937 cells induced with TNF-a.
PCR was performed with primers corresponding to the human IL-8 promoter.
Lanes 1-4: beads not pre-incubated with bridging antibody.
Lanes 5-8: beads pre-incubated with 5 µg bridging antibody.
Lanes 1, 2 & 5, 6: ChIP performed using anti-p65 mouse mAb, 2 µg per IP.
Lanes 3, 4 & 7, 8: ChIP performed using negative control mouse IgG.

For your convenience, Active Motif offers Ready-to-ChIP Chromatin from a number of ENCODE cell lines, which have been
optimally sheared by sonication and validated in ChIP. As a result,
you can more easily validate your own antibodies and primer
sets. The chromatin can be used with all of the ChIP-IT Kits andcontrols, so you can be certain that the only variable in validatinga new antibody for ChIP is the antibody itself.

Product

FormatCat. No.

Product

FormatCat. No.

Bridging Antibody for Mouse IgG

500 µg

53017

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

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11

Histone ELISAsHistone H3 phospho Ser28 & mono-, di- and tri-methyl Lys4 ELISA Kits
screen extracts for histone modifications
Active Motif has applied our histone-modification antibody
expertise to identify optimal antibody pairs for the detection of
specific histone modifications in a sandwich ELISA format. Now

The Histone ELISA advantage

it is easy to quickly screen extracts for changes in histone H3
phosphorylation and methylation levels.

FIGURE 1:
Histone H3 phospho Ser28 levels
in HeLa acid extracts.
78 ng to 5 µg untreated (copperline) and paclitaxel treated (purpleline) HeLa acid extract assayed with
the Histone H3 phospho Ser28
ELISA.

Screening extracts by standard immunoblotting methods is time
consuming. Our Histone Modification ELISA kits enable you toefficiently screen up to 96 wells at once, with little hands-ontime and colorimetric results in hours. Active Motif’s high-quality
histone antibodies guarantee specific results with extremely low
background (Figures 1 & 2) For added convenience, Paclitaxeltreated HeLa acid extract is included in the Histone H3 phospho
Ser28 ELISA Kit. Recombinant methylated histone H3 is includedin the methylation-specific ELISA kits.

Histone H3 phospho Ser28 (OD450 nm)

2.0

1.5

1.0

0.5

0.0

5

2.5

1.25

0.625

0.312

0.156

0.078

0

Acid Extract (µg)

2.5Histone H3
Histone H3 trimethyl Lys42

OD 450 (nm)Why Use Histone ELISA Kits?
• Sensitive – works with < 78 ng of acid extract

1.5
1• Specific – C-terminal histone H3 capture

0.5• Efficient – no time-consuming immunoblotting0• Convenient strip-well format – use only what you need

Histone H3 phospho Ser28

125

62.5

31.25

15.62

7.81

3.90

1.95

0

Recombinant Histone H3 (ng)FIGURE 2:
Histone H3 trimethyl Lys4 detection.
Recombinant Histone H3 and Recombinant Histone H3 trimethyl Lys4 wereassayed from 125 ng down to 1.95 ng using the Histone H3 trimethyl Lys4 ELISA.

Cell division is a complex, tightly regulated process that ismarked by mitosis. Two significant mitotic events include
microtubule spindle formation and chromosome condensation. Histone H3 is phosphorylated on serine 28 during mitotic
chromatin condensation before nuclear division occurs, whichmakes phosphorylation of histone H3 at Ser28 an important
marker for cells undergoing mitosis. With this simple ELISA
format, it is easy to assess levels of phosphorylated histone H3
Ser28 in cell extracts (Figure 1).

Histone H3 mono-, di- and tri-methyl Lys4
Methylation of Histone H3 at lysine 4 has been shown to indicate transcriptionally active chromatin. With the Histone H3
ELISA Kits for methylated Lys4, it is easy to screen for changes
in the degree of methylation at lysine 4 in your acid extract
samples (Figure 2). Histone H3 methylated Lys4 ELISA Kits areavailable for mono-, di- and tri-methylated lysine 4. Each kit
includes a recombinant Histone H3 mono-, di- or tri-methylated
lysine 4 (page 17) that can be used to quantitate the levels of
specific methylation in your sample. The ELISA kits utilize thesame histone H3 C-terminal antibody for capture, and each
kit uses either the mono-, di- or tri-methyl Lys4 antibody forspecific detection (Figure 3).

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FIGURE 3:
Specific histone H3 trimethyl Lys4 detection.
Recombinant Histone H3 monomethyl Lys4 (H3K4me1), Recombinant Histone H3
dimethyl Lys4 (H3K4me2) and Recombinant Histone H3 trimethyl Lys4 (H3K4me3)
were assayed from 80 ng to 1.25 ng with the Histone H3 trimethyl Lys4 ELISA. The
assay is clearly very specific for the trimethyl Lys4 modification on Histone H3.

Product

FormatCat. No.

Histone H3 phospho Ser28 ELISA

96 rxns

53100

Histone H3 monomethyl Lys4 ELISA

96 rxns

53101

Histone H3 dimethyl Lys4 ELISA

96 rxns

53102

Histone H3 trimethyl Lys4 ELISA

96 rxns

53103

HeLa acid extract

100 µg

36200

HeLa acid extract (Paclitaxel treated)

100 µg36201

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Chromatin Assemblyc h r o m at i n b i o l o g y

Chromatin Assembly Kit
generate high-quality chromatin for downstream success
Active Motif’s Chromatin Assembly Kit enables the generationof chromatin in vitro from linear or supercoiled DNA. Based on
an ATP-dependent method, the kit utilizes purified recombinanthuman chromatin assembly complex ACF and the histone
chaperone NAP-1 (h-NAP-1) with purified HeLa core histones forin vitro assembly of extended, regularly ordered, periodic arrays of
nucleosomes. The resulting chromatin closely resembles naturalin vivo chromatin, enabling studies of histone modifications andassociated proteins that are crucial to regulation of the target
DNA sequence.
The assembly of genomic DNA and histones into chromatin isa fundamental process that affects a broad range of genome-

dependent processes including DNA replication, DNA repair andgene expression. In general, there are ATP-dependent and ATP-independent methods for reconstituting or assembling chromatin in
vitro. The ATP-independent method results in a random arrangement of histones on the DNA that does not accurately reflect
the native core nucleosome. The resulting mononucleosomes are
not regularly ordered, extended nucleosomal arrays and the DNA
sequence used is usually no longer than 250 bp. To generate an extended array of ordered nucleosomes on a length of DNA greaterthan 250 bp, the chromatin must be assembled through the ATPdependent process. The Chromatin Assembly Kit provides an easy
and complete solution for ATP-dependent chromatin reconstitution and produces an excellent substrate for downstream assays.

The Chromatin Assembly Kit advantage
In the Chromatin Assembly method, recombinant h-NAP-1 andHeLa core histones are combined in a high-salt buffer, which
is ideal for proper histone configuration. Recombinant ACF
complex and sample DNA are then added with the complete
ATP regeneration system to complete the Chromatin Assembly.
After a four-hour incubation, the assembled chromatin is readyto use in downstream assays such as in vitro transcription assays,
chromatin immunoprecipitation and histone acetyltransferase(HAT) assays. Chromatin assembly is verified by partial digestion
with the provided Enzymatic Shearing Cocktail and analyzed bygel electrophoresis. High-quality chromatin should yield six ormore distinct bands (Figure 1). For your convenience, supercoiled
DNA is provided as a positive control.

Why use the Chromatin Assembly Kit?
• Generate chromatin from linear or supercoiled DNA

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) weredigested for 2 and 4 minutes, respectively, deproteinated, phenol/chloroform extracted and run onan agarose gel. Each sample type processed with the
Chromatin Assembly Kit resulted in regularly spaced
nucleosomes.

• ATP-dependent method results in an extended array ofregularly spaced nucleosomes• Excellent substrate for gene regulation experiments• Easy protocol, simply incubate the supplied components
with your DNA

www.activemotif.com

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Product

FormatCat. No.

Chromatin Assembly Kit

10 rxns

53500

EUROPE +32 (0)2 653 0001

NORTH AMERICA 877 222 9543

13

Histone Purification
Histone Purification Kit
isolate pure fractions of core histone proteins while preserving post-translational modifications
Now you can easily purify histones and further separate thefractions of core histones from any cell culture or tissue sample

while maintaining post-translational modifications like acetylation, methylation and phosphorylation states.

How does it work?
Active Motif’s Histone Purification Kit enables you to isolate core
histones from any cell culture or tissue sample (Figure 1). Unlike
histone purification by acid precipitation, our method utilizes
a unique purification resin and a series of proprietary elution
buffers to isolate very pure histone fractions. The resin has a highbinding capacity for histones, so core histones can be isolated
from small cell culture samples on up to grams of tissue.

Acetylation

Methylation

Sequential elution steps let you collect the core histones as one
total population containing H2A, H2B, H3 and H4, or further separated into two populations: one enriched for H2A/H2B dimers
and a second fraction containing > 90% pure H3/H4 tetramers.

Phosphorylation

FIGURE 2:
Post-translational modifications preserved.
Acetylation, methylation and phosphorylation states are preserved as wellor better with the Histone Purification Kit compared with standard acidprecipitation method.

Better substrate for downstream assays
Core histones isolated by the Histone Purification Kit method
are highly pure and suitable substrates for downstream assays.
Purified histones can be used with the Chromatin Assembly Kit(page 13) to enable the generation of chromatin that very closely
resembles native chromatin for functional assays (Figure 3).

FIGURE 1:
SDS-PAGE of histone fractions purified using the Histone Purification Kit.
Ten µg of sample were loaded per lane and run on a 16% Tris-glycine gel. Leftpanel: core histones purified from logarithmically growing tissue culture cells(TC) and core histones isolated from rat brain tissue (Brain). Right panel: H2A/
H2B (Lane 1) and H3/H4 (Lane 2) fractions purified from HeLa cells.

FIGURE 3:
Chromatin assembled with purified histones.
Histones were purified from HeLa cells and usedin the Chromatin Assembly Kit. The ordered spacing of nucleosomes was confirmed and analyzed
by agarose gel.

Preserve important modifications
Preserving post-translational modifications like acetylation,
methylation and phosphorylation is critical when investigating
the role of histones in transcription or chromatin biology.
Our proprietary buffer system is optimized to maintain thesemodifications while separating the histones into pure fractions(Figure 2).

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Product

FormatCat. No.

Histone Purification Kit

10 rxns

40025

JAPAN +81 (0)3 5225 3638

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Histone Purificationc h r o m at i n b i o l o g yHistone Purification Mini Kit (Spin Column Format)
faster, easier histone purification while preserving post-translational modifications
Active Motif first brought you the ground breaking Histone
Purification Kit, enabling the fast and easy isolation of core
histones from any cell culture or tissue sample. Now, we are
expanding upon that innovative product by introducing our new

spin column-based Histone Purification Mini Kit, which is evenfaster, more sensitive and easier to use. Purification of histone
from yeast cells is also possible with this new kit. And, like theoriginal kit, post-translational modifications remain intact.
FIGURE 1:
Histone purification in a new spincolumn format.
Convenient spin column-based
protocol enables faster purificationof histones from multiple samples
simultaneously.

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

How does it work?
Our unique method utilizes a purification column with a proprietary binding matrix (Figure 1) and a series of proprietary buffers
to isolate very pure fractions of histones. Using the Histone
Purification Mini Kit, core histones can be 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 highaffinity for histones, so histones can be purified from as little as8x105 cells. While you can not purify the same amount of histones as with the Histone Purification Kit, spin columns make iteasier to isolate histones from multiple samples simultaneously.
SourceYield

Adherent Cells

0.1 mg total core histones from 8 x 106 cells.

Suspension Cells

0.1 mg total core histones from 8 x 106 cells.

Yeast

0.5 mg total core histones from 8 x 107 cells.

Tissue1 mg histone per gram of tissue*

TA B L E 1 :Representative histone yields.
Yields are approximate. Results may vary according to cell or tissue type.

Post-translational modifications remain intact
Preserving post-translational modifications like acetylation,
methylation and phosphorylation is critical when investigating the role of histones in transcription or chromatin biology.
Our proprietary buffer system is optimized to maintain thesemodifications while separating the histones into pure fractions(Figure 2). Histones isolated with the Histone Purification Mini
Kit are highly pure and are suitable as substrates in many downstream assays. Purified histones can be analyzed by Western
blot or mass spectrometry, or assembled into chromatin thatclosely resembles native chromatin for functional assays using
the Chromatin Assembly Kit (see page 13).

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FIGURE 2:
Analysis of histone fractions by SDS-PAGE and Western blot.
Histones were purified from a variety of sources using the Histone Purification
Mini Kit. (Unstim.: unstimulated HeLa cells, Butyrate: sodium butyrate-treated
HeLa cells, Paclitaxel: paclitaxel-treated HeLa cells, Tissue: rat brain tissue, Yeast:
budding yeast). The first and second elutions (labeled 1st and 2nd) from eachsample were analyzed on a 16% Tris-glycine SDS-PAGE gel (top panel) and byWestern blot (bottom panel). Antibodies recognizing acetyl-histone H3 (Ac-H3:
Catalog No. 39139, 1:500 dilution), phospho-Ser28 histone H3 (Phos-H3: Catalog
No. 39098, 1:1,000 dilution), and trimethyl-Lys4 histone H3 (Me-H3: Catalog No.
39159, 1:1,000 dilution). The migration of the histones in the SDS gel is indicatedby the bracket to the left of the gel.

Product

FormatCat. No.

Histone Purification Mini Kit

20 rxns

40026

EUROPE +32 (0)2 653 0001

NORTH AMERICA 877 222 9543

15

Histone Acetylation & Deacetylation
HAT & HDAC Assay Kits
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 activity of histone
acetyltransferases and histone deacetylases in your cell andnuclear extracts, immunoprecipitates and purified enzymes, aswell 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.

The HAT Family
Histone acetyltransferases (HAT) are enzymes that acetylate
conserved lysine amino acids on histones. Generally, histone
acetylation is associated with the 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 of gene expression, their activity is much studied, as are compounds that inhibit
HAT and HDAC activity.

How does the HAT Assay Kit work?
Assaying HAT activity is easy with this 96-well plate format. Simply incubate your HAT with your choice of the provided 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 thehistone acetyltransferase activity to give a fluorescent readout.
This makes it easy to generate standard curves with acetyl-CoAor b-mercaptoethanol, so you can relate the fluorescence ofyour HAT to pmol/min/µg specific activity.

FIGURE 1:
HAT inhibitor effects on p300 activity.
50 ng p300 were assayed per well with 50 µM acetyl-CoA 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.

Active recombinant p300 protein is provided as a control for use
with your samples; enough is provided to use as a HAT to screen
an entire 96-well plate of inhibitors. Anacardic acid is also provided for use as a control, as it is a potent HAT inhibitor (Figure 1).

How do the HDAC Assay Kits work?
The HDAC Assay Kits utilize a peptide substrate that contains anacetylated lysine residue that can be deacetylated by 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 thesubstrate is deacetylated, the lysine reacts with the Developing
Solution and releases either the chromophore or the fluorophore from the substrate, which produces either a colorimetricor fluorescent product. The colorimetric product absorbs maximally at 405 nm; the fluorescent product can be read with an
excitation wavelength of 360 nm and emission wavelength of
460 nm (Figure 2).

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FIGURE 2:
HDAC activity in HeLa cells.
HeLa nuclear extracts were assayed at 0 to 10 µg per well using the fluorescentversion of the HDAC Assay Kit. Untreated extract results are shown with apurple line, and extracts inhibited with 1 mM Trichostatin A are shown with acopper line.

Product

FormatCat. 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

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Recombinant Histones

c h r o m at i n b i o l o g y

Recombinant Methylated Histones
new with site- & degree-specific methylated lysines
Active Motif is pleased to announce the first release in its newcollection 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 specificlysine methylations influence nucleosome remodeling and thebinding of associated chromatin proteins.1

Histones & chromatin structure

FIGURE 1:
Ordered spacing of nucleosomes after
enzymatic digestion of assembled chromatin.
4.5 µg Recombinant Histone H3 dimethyl Lys9
was used to generate chromatin in vitro using theChromatin Assembly Kit (see page 13). One µg ofassembled chromatin was digested for 2 minutes(lane 2) and 4 minutes (lane 3). Lane 1 is 100 bp marker.

Methyl-lysine residues in nucleosomal histones are thought tomediate interactions with the protein complexes involved inregulating transcription, replication and DNA repair. In order toinvestigate these complex functional questions, histones withspecific methylation states are required to evaluate which methylation patterns are key to regulatory processes. Active Motif ispleased to be the first to offer methylated histone H3 proteinsthat can be used in nucleosome remodeling assays to investigate
the implication of specific methylation on chromatin function.
1

Methylated histone H3
Active Motif currently offers recombinant histone H3 proteinsthat are mono-, di- and tri-methylated at lysines 4, 9 & 27. Visit
our website to see the latest releases of methylated histones.

2

3

Anti-Histone H3 K9me1
H3

Anti-Histone H3 K9me2

H3
H3
H3
K9me1 K9me2 K9me3

H3

Anti-Histone H3 K9me3

H3
H3
H3
K9me1 K9me2 K9me3

H336

How is the methylation state made?

36

36

Recombinant methylated histones are created via a chemical
alkylation reaction* that introduces an analog of methyl lysine.
This specific chemical treatment enables the site and degreeof methylation 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 recombinanthistones are perfect for any functional assay.

30

30

30

16

16

Product

FormatCat. 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

16

H3
H3
H3
K9me1 K9me2 K9me3

6

6
6

1

2

3

4

5

6

7

8

9

10

11

12

FIGURE 2:
Analysis of mono-, di- and tri-methylated Recombinant Histone H3.
Western blot analysis of 1 µg Recombinant Histone H3 (lanes 1, 5 & 9),
1 µg Recombinant Histone H3 monomethyl Lys 9 (lanes 2, 6 & 10),
1 µg Recombinant Histone H3 dimethyl Lys9 (lanes 3, 7 & 11) and1 µg Recombinant Histone H3 trimethyl Lys9 (lanes 4, 8, and 12) probed with
Histone H3 monomethyl Lys9 pAb (Cat. No. 39249) (lanes 1-4),
Histone H3 dimethyl Lys9 pAb (Cat. No. 39239) (lanes 5-8) andHistone H3 trimethyl Lys9 pAb (Cat. No. 39161) (lanes 9-12).

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

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17

DNA Methylation
MethylDetector™
simplified bisulfite conversion of DNA with easily verified results
Active Motif’s MethylDetector™ Bisulfite Modification Kit simplifies analysis of DNA methylation. It comes complete with optimized reagents for performing DNA conversion with bisulfite, plus
time-saving DNA purification columns and positive control PCR
primers to validate your results.
DNA methylation is a naturally occurring event that affects cellfunction by altering gene expression. A methyl group is added to
the fifth-carbon of cytosine in a CpG dinucleotide by DNA methyltransferase. As aberrant methylation is prevalent in many humancancers, and because methylation is also involved in embryonicdevelopment and cell cycle regulation, much research depends on
accurately quantifying DNA methylation. Many DNA methylation
analysis methods begin by using bisulfite to convert unmethylated
cytosines to uracils.1, 2 Unmethylated cytosine is changed to uracil,

which base pairs with adenosine, not guanosine. During conversion, methylated cytosines remain unchanged. The DNA is thenamplified by PCR and analyzed by sequencing or restriction digest.
A methylation profile of the sample can then be created by comparing the sequence of the converted DNA to untreated DNA.
However, bisulfite conversion can be technically challenging, and
it is desirable to confirm that the process was successful before
spending time and money on sample analysis. To help ensure yoursuccess, the MethylDetector Kit provides optimized conversion
reagents, an easy-to-use protocol and positive control PCR primers that are specific for bisulfite-converted DNA. Because theseprimers produce a PCR product only if conversion has occurred,
you can confirm the procedure worked before performing
sequencing or other analysis methods.

The MethylDetector™ advantage
In the MethylDetector method, DNA of interest is rapidly heat
denatured in a thermocycler in the presence of the bisulfite
conversion reagent. The temperature is then lowered and theconversion reaction is performed. Unlike other methods, MethylDetector does not require a separate denaturation step as theconversion reagent includes a DNA denaturant, saving you timeand effort. After DNA conversion, the sample is added to theincluded DNA purification columns, and a simple, on-column
desulfonation reaction is performed. Ready-to-use DNA is theneluted from the columns. For your convenience, the included
positive control PCR primers can be used to assess the successof the bisulfite conversion before you spend time and money on
DNA sequencing. The included primers only anneal to converted
human DNA (Figure 1).

FIGURE 1:
Agarose gel analysis of PCR
products generated withMethylDetector.
Three different DNA conversions were
performed (Lanes 1-3) and compared
to an unconverted DNA control(Lane 5) and to a no DNA control(Lane 4). The presence of PCR productin only the converted samples demonstrates the conversion efficiency andreproducibility of MethylDetector.

Why use MethylDetector™?
• Works efficiently with high G/C content sequences anduncut DNA

Product

FormatMethylDetector™

50 rxns

55001• Reproducible assay consistently provides 99% conversion
efficiency of unmethylated cytosines

Fully Methylated Jurkat DNA

10 µg

55003

• Optimized reagents and protocol with proven human controls• Combined thermal denaturation and conversion reaction
eliminates NaOH-mediated denaturation and streamlines
procedureCat. No.

REFERENCES
1. Frommer, M. et al. (1992) PNAS 89: 1827.
2. Clark, S.J. et al. (1994) Nuc. Acids Res. 22: 2990-2997.

• DNA purification columns eliminate the need for
precipitation and a separate desulfonation step
• High yield of converted DNA ideal for downstream analysis

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DNA Methylationc h r o m at i n b i o l o g y

MethylCollector™
rapid and efficient comparison of methylation in various samples
Active Motif’s MethylCollector™ Kit provides
users with a fast and efficient method* forisolating and comparing CpG-methylated DNA
from various cell or tissue samples. MethylCollector uses a recombinant Methyl-binding
protein (MBD2b) to capture DNA, rather than
traditional antibody-based immunoprecipitations, improving sensitivity.

MethylCollector™ advantages• Fast and easy protocol completed inless than 4 hours• Flexible – enables detection from
5 ng to 1 µg of DNA• Suitable for use with DNA fragmented bysonication or enzymatic digestion• Positive control DNA and PCR primers
help ensure your success

The MethylCollector™ method
In the MethylCollector method, His-tagged
recombinant MBD2b protein specifically binds
to CpG-methylated DNA fragments that havebeen prepared by enzymatic digestion orsonication. These protein-DNA complexes arecaptured with nickel-coated magnetic beads
and subsequent wash steps are performed witha stringent high-salt buffer to remove DNA
fragments that have little or no methylation.
Ready-to-use methylated DNA is then eluted
from the beads (Figure 1). MethylCollector ishighly efficient, enabling analysis of themethylation state of any specific locus on
genomic DNA isolated from less than 800 cells(~5 ng DNA, Figure 2).

Applications of MethylCollector™
The highly specific isolation of methylated DNA
by MethylCollector enables powerful applications, including rapid screening of the methylation status of multiple loci in tumor tissue orcells. It can also be used to detect changes in
DNA methylation in other situations, e.g.
cellular differentiation, aging and cancer.

FIGURE 1:
Flow chart of the MethylCollector process.
In a MethylCollector assay, genomic DNA of interest is sheared by either enzymatic digestion orsonication. The sheared DNA is then incubated with His-tagged recombinant MBD2b protein, which
has a strong affinity for CpG-methylated DNA. These protein-DNA complexes are captured withnickel-coated magnetic beads and stringent washes are performed to remove DNA fragments thathave little or no methylation. The methylated DNA is then eluted from the beads and PCR is performed on the resulting supernatant, using primers that are specific to amplify the locus of interest.

FIGURE 2:
Comparison of CpG-methylated DNA
isolated from HeLa and A-431 cells
using MethylCollector.
Genomic DNA from A-431 (positive
control DNA) and HeLa (negative
control DNA) cells were enzymaticallydigested by Mse I for 2 hours. Increasing amounts of fragmented DNA (from5, 40 and 100 ng) were then incubated for 1 hour with 1 µg His-MBD2b recombinant protein in thepresence of nickel-coated magnetic beads. After washing, CpG-methylated complexes were eluted.
The isolated DNA was then analyzed by 36 cycles of PCR using the kit’s control primers, which amplify a locus that is not methylated in HeLa but highly methylated in A-431. PCR on MethylCollectorenriched A-431 DNA generates robust signals that are proportionate to the amount of DNA starting
material (left panel, lanes 4 to 6). No signal is observed with enriched HeLa DNA (right panel, lanes
4 to 6) or in those samples where His-MBD2b protein was omitted from the binding reaction (leftand right panels, lanes 1 to 3). Taken together, these results indicate that MethylCollector specificallyenriches for methylated DNA fragments, and that this enrichment is due to the presence of the kit’s
His-MBD2b protein.

Product

FormatCat. No.

MethylCollector™

25 rxns

55002*Technology covered under U.S. Patent No. 7,425,415.

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19

Nuclear, Whole-cell & Cytoplasmic Extraction
Nuclear Extract Kit
high yields of specifically segregated extracts
The Nuclear Extract Kit is ideal for the preparation of nuclear,whole-cell and cytoplasmic extracts from mammalian cells andtissues. The resultant high-quality extracts may be used with
Active Motif’s TransAM™ Kits or in gelshift assays, Western blots,
DNA footprinting or as a starting point for transcription factor
purification.

The Nuclear Extract Kit eliminates the need to optimize reagentsand ensures consistently high yields. The detailed protocol helps
ensure that your extract is not contaminated with proteins fromother cellular compartments (Figure 1).

The Nuclear Extract Kit advantage
In the Nuclear Extract Kit, cells are collected in ice-cold PBS in
the presence of phosphatase inhibitors to limit further protein
modifications. Next, the cells are resuspended in hypotonic
buffer to swell the cell membrane. Addition of detergent causes
leakage of the cytoplasmic proteins into the supernatant. After
collection of the cytoplasmic fraction, the nuclei are lysed andthe nuclear proteins are solubilized in lysis buffer in the presence of the protease inhibitors. Whole-cell extracts can also be
prepared by collecting the cells in the PBS/phosphatase inhibitors solution and lysing in lysis buffer. Solubilized proteins are
separated from the cell debris by centrifugation. The concentration of protein in the cell extract is then measured by Active
Motif’s ProStain Protein Quantification Kit (Cat. No. 15001) orwith a Bradford-based assay.

• Complete kit contains all required reagents

FIGURE 1:
Specific extraction of nuclear and cytoplasmic extracts.
Nuclear and cytoplasmic extracts were prepared using the Nuclear Extract Kit
from HeLa samples unstimulated or stimulated with TNF-a for 30 minutes andassayed using the TransAM™ NFkB p50 Kit. Because activated NFkB translocatesto the nucleus, only nuclear extract from stimulated cells should contain activated NFkB.

• Prepare nuclear, cytoplasmic or whole-cell extracts withone kit

Product

FormatCat. No.

Nuclear Extract Kit

100 rxns
400 rxns

40010
40410

Why use the Nuclear Extract Kit?
• Quality-controlled reagents ensure reproducibility• No need to optimize your own procedure• Ability to prepare extracts from both cultured cells andtissue samples

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Co-Immunoprecipitationc h r o m at i n b i o l o g y

Universal Magnetic Co-IP Kit
lower background; suitable for both nuclear & whole-cell complexes
The Universal Magnetic Co-IP Kit improves co-immunoprecipitation (Co-IP) through the use of protein G-coated magnetic beads,
which speed and simplify the IP and wash steps while greatly
reducing background. The kit includes optimized reagents formaking both nuclear & whole-cell extracts from cells or tissue,
giving you the flexibility to Co-IP any protein complex, whether
it was originally bound to DNA (Figure 1) or in the cytoplasm.

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
reagents that 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 theDNA, so they are intact and ready for Co-IP.

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

Universal Magnetic Co-IP Kit advantages

FIGURE 2:
Detection of acetylated Histone H3.
HeLa nuclear extracts were made using
the Universal Magnetic Co-IP Kit anda 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). The pan acetyl-H3(arrow) was detected only in samples
made using the kit’s gentle nuclear
extraction procedure.

• Magnetic beads simplify procedure and reduce background• Optimized extraction method maintains nuclear protein
complexes• Preserve protein modifications

Simpler procedure, lower background
The Universal Magnetic Co-IP Kit utilizes protein G-coated magnetic beads, which simplify Co-IP by enabling the IP and wash
steps to be performed in seconds, rather than having to use
centrifugation. Because these beads have very low non-specificbinding, background is reduced even while using the kit’s low-salt
Co-IP/Wash Buffer, which helps maintain weaker complexes.

FIGURE 3:
Preserve acetylation and methylation.
Nuclear extracts were made from HeLa
cells treated with 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 ten µg 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 thesample made using the kit. 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 wholecell extraction reagents, so you can perform IP on all types ofprotein complexes. The kit also includes protein G-coated magnetic beads, a unique Co-IP/Wash Buffer as well as phosphatase,protease and deacetylase inhibitors that preserve the integrity ofthe proteins and protein modifications (Figures 2 & 3). Finally, thekit includes a strong bar magnet, so you can take advantage ofthe improved wash and IP steps enabled by the magnetic beads.
This makes the Universal Magnetic Co-IP Kit a simple, flexible
and complete solution for getting more from your Co-IP.

www.activemotif.com

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Product

FormatCat. No.

Universal Magnetic Co-IP Kit

25 rxns

54002

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DNA Damage and Repair
DNA Damage Assay
simple and fast 2-color fluorescent assay of H2AX phosphorylation
When cells are exposed to ionizing radiation, double-strand DNA
breaks occur that stimulate the phosphorylation of the histone
variant H2AX at serine 139 by the ATM kinase. As H2AX is present
at approximately 10% of the level of histone H2A, detection ofphosphorylated H2AX is an extremely sensitive marker to assess
the effects and timing of DNA damage-inducing agents.

Active Motif’s DNA Damage Assay makes screening compounds
for their ability to induce DNA damage or apoptosis sensitive,
accurate and reproducible. This assay can also be used to detect
cells committed to undergo programmed cell death, as chromatin
is cleaved by caspase-dependent nucleases, also leading to H2AX
phosphorylation.

How does the assay work?

FIGURE 2:
Etoposide-induced DNA
damage in HeLa cells.
Levels of phospho-H2AX measured in untreated (control)and cells treated with etoposide. Averages of quadruplicates are shown.

The DNA Damage Assay is a cell-based assay conducted in
96-well plates. Each kit includes all reagents necessary for two
96-well plates. Cells are grown and treated in the plate, fixed,
then incubated with the phospho-histone H2AX (Ser 139) antibody. Next, a short incubation with Chromeo™ 488-conjugatedsecondary antibody is performed, followed by a readout on astandard or high-content fluorescence plate reader.

High specificity and low background
The DNA Damage Assay relies on a high-quality rabbit polyclonal
phospho-histone H2AX (Ser 139) antibody that is subsequently
detected with the Chromeo 488-conjugated Goat anti-Rabbit
secondary. Our Chromeo fluorescent secondaries are highly specific with extremely low background, so your results are always
accurate (Figure 1).
The assay includes a control compound, etoposide, which
induces high levels of DNA damage and apoptosis (Figure 2). The
phospho-histone H2AX (Ser 139) antibody is extremely specific,
resulting in very low background in the assay. Propidium iodide
is included to provide a marker for total cell count. This simple
detection method utilizing Chromeo 488 fluorescence and propidium iodide stain makes the kit ideal for both high-throughput
and high-content screening in all mammalian cell types (Figure 3).
FIGURE 1:
Merged image of etoposidetreated HeLa cells.
HeLa cells stained only withpropidium iodide (PI) appear
red, while those stained withboth PI and phospho-H2AX
detected with Chromeo 488
appear more yellow.

REFERENCE
1. Rogaku, E. P. et al. (1992) J. Biol.
Chem. 273(10): 5858-5868.

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FIGURE 3:
Single cell analysis of phospho-H2AX in HeLa cells by the IsoCyte™ scanner.
Untreated HeLa cells (upper panel) are detected mostly in the propidium iodide
channel while etoposide-treated cells are seen in the green (488 nm) channel,
indicating a high degree of DNA damage.

Product

FormatCat. No.

DNA Damage Assay (Fluorescent)2 x 96 rxns

18030

Histone H2AX phospho Ser 139 pAb

200 µl39117

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Mitosis Determinationc h r o m at i n b i o l o g y

Mitotic Index Kits
quickly determine the proportion of cells in a population undergoing mitosis
Cell division is a complex and tightly regulated process, the final
step being mitosis. The ability to detect cells undergoing mitosis
is a simple way to characterize the effect of drug treatments oncell division. During the progression of interphase and G2 into M
phase, chromosomes are condensed to allow nuclear division tocontinue. During this event, histones are subject to post-transla-

tional modifications in a specific and temporally regulated manner.
Studies have shown that histone H3 is phosphorylated at serine 28(and several other sites) during mitosis. Therefore, the phosphorylation of histone H3 at serine 28 is a useful marker for mitosis,
and this specific phosphorylation event can be used to determine
quickly and accurately the mitotic index of a population of cells.

The Mitotic Index Kit advantage
The Mitotic Index Assay Kit is a simple and accurate method todetermine the percentage of cells undergoing mitosis within apopulation. The kit uses a monoclonal antibody (clone HTA28)
that is specific for phosphorylation at serine 28 of histone H3
and an exceptionally bright fluorescent secondary antibody,
Chromeo™ 488 Goat anti-Rat IgG, for detection. Propidium
iodide is used to stain all cells, and the mitotic index is determined by calculating the percentage of cells undergoing mitosis
within the total population (Figure 1). For added convenience, themicrotubule poison paclitaxel is included in the kit to arrest cells
in mitosis, providing a high mitotic-index reference population(Figure 2).

FIGURE 1:
HeLa cells fixed with methanol and assayed with the Mitotic Index Assay Kit.
Phospho-histone H3 (Ser28) detected with Chromeo 488 Goat anti-Rat IgG isshown in the first image. Nuclear staining of all cells with propidium iodide isshown in the center image. The merged image is shown on the right; cells withboth green Chromeo 488 signal and red propidium iodide signals appear yellow.

Why use the Mitotic Index Kit?
• Highly specific monoclonal phospho-Histone H3 (Ser28)
clone HTA28• Exceptionally bright Chromeo™ 488 detection• High-throughput compatible method that is easily adaptable to any plate format• Whole cell analysis with no cell lysis steps• Available in chemiluminescent and colorimetric formats

Please browse our complete line of
Chromeo-labeled fluorescent antibodies at:www.activemotif.com/chromeo.

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FIGURE 2:
Whole well analysis of paclitaxel-treated vs. untreated HeLa cells.
HeLa cells were treated with 1 µM paclitaxel diluted in complete medium for6 hours prior to analysis using the Mitotic Index Assay Kit.

Product

FormatCat. No.

Mitotic Index Assay Kit

5 x 96 rxns

18020

Mitotic Assay Kit (Color)

2 x 96 rxns

18021

Mitotic Assay Kit (Chemi)

2 x 96 rxns

18022

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Biotech Line A/S
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Active Motif Offices

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JAPAN
Azuma Bldg. 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
Email: japantech@activemotif.com

U.S.
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008 USA
Toll Free: 1 877 222 9543
Direct: 1 760 431 1263
Fax: 1 760 431 1351
Email: sales@activemotif.com
EUROPE
104 Avenue Franklin Roosevelt – Box 25
B-1330 Rixensart, Belgium
Germany Free Phone: 0800/181 99 10
France Free Phone: 0800/90 99 79
UK Free Phone: 0800/169 31 47
Other countries, Direct: +32 (0)2 653 0001
Fax: +32 (0)2 653 0050
Email: eurotech@activemotif.com
JAPAN
Azuma Bldg. 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
Email: japantech@activemotif.com

1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
USA

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