Histone Analysis

Histone Analysis
Histone Antibodies
Histone Purification
Recombinant Histones
Histone Modifying Enzymes
Histone Modification ELISAsHistone Demethylase Assay
Histone Peptide Arrays
Histone Binding Assay
HAT/HDAC Assays
Chromatin Assembly

innovative tools designed to help unravel the histone code

Active Motif’s unique portfolio of histone technologies provides
researchers with a complete solution for the analysis of histones
and their post-translational modifications, beginning with histone
purification and continuing through to chromatin assembly.
Let Active Motif’s antibodies, enzymes and modification-specificassays simplify your histone analysis.

Histone Analysis – tools designed to help unravel the histone code
Histone modifications such as acetylation, phosphorylation and methylation at specific amino acid residues on
the histone globular domain and the N-terminal tails have been shown to influence and regulate transcription,
chromosome packaging and DNA damage repair. Due to the importance of histone modifications in regulating
chromatin structure and disease, Active Motif has developed a variety of products that simplify histone analysis.
Histone Purification
Active Motif’s Histone Purification andHistone Purification Mini Kits enable you toisolate core histones from any cell culture
or tissue sample while preserving their posttranslational modifications. The kits use aunique purification resin and a series ofproprietary elution buffers to isolate very
pure fractions of histones (Figure 1).
Unlike standard acid extraction techniques, it
is possible to isolate core histones as eithera single fraction, or to further separate theminto H2A/H2B and H3/H4 fractions (Table 1).
Additionally, our unique extraction buffer
prevents unwanted enzymatic reactions fromoccurring, thereby preserving the existing
histone modifications.
Purified histones are ready for downstream
analysis with Active Motif’s many different
histone modification antibodies, or they canbe used as substrates in functional assays,
such as Active Motif’s Histone Modification
ELISAs and Chromatin Assembly Kits.

MW

Log.

Mitotic
ArrestedMW

SodiumButyrate

98

98

64

64

50

50

36

36

30

30

• Preserves modifications on histones,
such as acetylation, phosphorylationand methylation• Purifies total core histones (H2A, H2B,
H3 & H4) or separate fractions of
H2A/H2B dimers and H3/H4 tetramers• Histone Purification Mini Kit can purify
histones from budding yeast

Core Histones

98
64
50
36

H3
H2B
H2A
H4

16

6

30

H3

16

H2B
H2A

H3
H2A/H2B

16

H4

H4

6
6

4

4

Figure 1: Core histone purified from HeLa cells and brain tissue.
Ten µg of sample were loaded per lane. Left: Core histones purified from logarithmically growing mitotic arrested
or sodium butyrate-treated HeLa cells. Center: Separate H2A/H2B and H3/H4 fractions purified from HeLa cells.
Right: Core histones isolated from rat brain tissue.

Kit

FormatElutionCapacity

Histone Purification Kit

Gravity Flow

Separate H2A/H2B &H3/H4 fractions

05-2.5 mg

Spin Column

H2A, H2B, H3 & H4 ina single fraction

0.5-2.5 mg

Mini Spin Column

H2A, H2B, H3 & H4 ina single fraction

0.1-0.5 mg

Histone Purification Mini Kit

Advantages

MW

Table 1: Comparison of the original Histone Purification Kit and the Histone Purification Mini Kit.

To learn more about either the Histone Purification Kit or the Histone Purification Mini
Kit, please call or visit us at www.activemotif.com/histonepur.
Product

FormatCatalog No.

Histone Purification Kit

10 rxns

40025

Histone Purification Mini Kit

20 rxns

40026

On the cover: Ribbon diagram of the nucleosome core particle structure (H2A.Z nucleosome, pdb entry 1F66) viewed down the superhelical axis (left) and rotated 90° (right).
Original figure was prepared by Dr. Karolin Luger, Department of Biochemistry and Molecular Biology, Colorado State University.

www.activemotif.com

MODified™ Histone Peptide Array & Analysis Software
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This unique histone array contains up to four
modifications per 19mer peptide to study
not only individual modifications, but also to
determine if neighboring modifications altersite recognition and binding. The MODified
Histone Peptide Array can be used to screen
antibodies for cross-reactivity or to study
protein and enzyme interactions (Figure 3).
The simple array protocol works like a
Western blot. Either ECL-based or colorimetric detection systems can be used. The image
is captured using film or a CCD camera; no
special equipment is needed.
The MODified Histone Peptide Arrays areavailable individually, or in packs of five. For
a complete solution, the MODified Array
Labeling Kit contains the necessary buffers
and reagents for ECL-based detection.

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The MODified™ Histone Peptide Array* is avaluable research tool that can be used toscreen antibodies, proteins and enzymes forinteractions with histones and their posttranslational modifications. Each array contains 384 different histone modification combinations in duplicate. Modifications include
acetylation, methylation, phosphorylationand citrullination on the N-terminal tails ofhistones H2A, H2B, H3 and H4.

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Figure 2: ECL detection and graphical analysis of the cross-reactivity of Histone H3 trimethyl Lys9 antibody.
Active Motif’s Histone H3 trimethyl Lys9 (H3K9me3) pAb (Catalog No. 39161) was used at a 1:2,000 dilution on the
MODified Histone Peptide Array. Anti-rabbit HRP secondary antibody was used at a 1:2,500 dilution, followed by ECL
detection and image capture with a CCD camera. Active Motif’s Array Analyse Software was used to analyze spot
intensity and generate a graphical analysis of decreasing specificity factors, which is the ratio of the average intensityof all spots containing H3K9me3 divided by the average intensity of all spots not containing H3K9me3. The results
show this antibody is very specific for histone H3 trimethyl Lys9, with little cross-reactivity for other modifications.

Advantages• Histone specific – unique array panel
tests for specific histone modifications• Study neighboring effects – each
peptide contains up to four modification combinations, enabling analysis of
the effects of neighboring modifications• Detects like a Western blot – fastand easy to use; works with either
ECL-based or colorimetric detection

To learn more about the MODified™ Histone Peptide Arrays, or to download the free
Array Analyse Software, please visit www.activemotif.com/modified.

Free Software for Analysis
Active Motif’s Array Analyse Software isa free program designed for use with the
MODified Histone Peptide Arrays. This PC
compatible software will analyze the spot
intensities from the MODified array and generate a graphical analysis of the histone modification interactions (Figure 2). Information
about spot intensity, averages and errors canbe saved in Excel-compatible files. For added
convenience, up to three individual modifications can be displayed in superposition to
the experimental data, enabling better
visualization of neighboring effects.

Histone Antibodies
Active Motif offers a variety of antibodies
to histones and biologically relevant histone
modifications. Each antibody has been
rigorously tested and validated for use inimportant applications such as immunofluorescence (IF), Western blotting and chromatin
immunoprecipitation (ChIP). Our years ofexpertise in antibody development ensuresthat only the highest quality antibodies areoffered for use in your research.

• Immunogen selection – ensures theantibody recognizes the modification ofinterest, and does not cross-react withrelated proteins• Specificity screening – every antibody
must have a greater than 25-foldselectivity for the desired modification• Application validation – important
applications are tested, giving you confidence when using them in your research

To see our extensive list of histone
modification antibodies, including quality
control data for IF, Western blot, ChIP or
MODified Histone Peptide Array, please
visit www.activemotif.com/histoneabs.
To learn about our fluorescent antibodies
for IF, or our antibody labeling kits, visit
www.activemotif.com/chromeo.

*CelluSpots™ arrays are manufactured under license by INTAVIS Bioanalytical Instruments AG and sold through Active Motif as MODified™ Histone Peptide Arrays

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Histone Analysis – tools designed to help unravel the histone code
Histone Modifying Enzymes
Histones are subject to a variety of posttranslational modifications that influencea number of nuclear processes. To better
investigate some of the complex functionalquestions about chromatin-associated proteins, nucleosome remodeling, transcriptional
regulation, replication and DNA repair, Active
Motif offers a range of histone modifying
enzymes for the following protein types:• Methyltransferases• Demethylases• Acetyltransferases• Deacetylases

These modifying enzymes can be used inconjunction with Active Motif’s antibodies,recombinant histones, ELISAs and MODified™
Histone Peptide Arrays (Figure 3).
Figure 3: Images of ECL detection of MODified
Histone Peptide Arrays treated with G9a.
MODified Histone Peptide Arrays were treated withA) 25 µM G9a methyltransferase (Catalog No. 31327),
B) 25 µM G9a mutant H904K (Catalog No. 31328), orC) no enzyme control, overnight in the presence of
1 mM AdoMet. The arrays were detected using aHistone H3 dimethyl Lys9 antibody. Novel methylation sites were observed on the array treated withwild-type G9a histone methyltransferase, showing
the activity of this histone modifying enzyme on the
peptide substrates.

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To view our full listing of histone modifying enzymes and their associatedtechnical data sheets, please visit www.activemotif.com/hismodenz.

Formaldehyde Detection of Histone Demethylase Activity
The Histone Demethylase Assay Kit canbe used to analyze the overall conversion
efficiency of an LSD1 sample, or to screen
compounds that cause changes in histone
demethylation activity.

Product

FormatCatalog No.

Histone Demethylase Assay (Fluorescent)

48 rxns

53200

Recombinant LSD1 protein, active

50 µg

31334

As shown in Figure 4, the LSD1 enzyme is able
to more efficiently demethylate the included
recombinant histone H3K4me2 protein than ahistone H3K4me2 peptide substrate. Because
the recombinant histone more closely
resembles a native histone, the Histone
Demethylase Assay enables more accurate
analysis of histone demethylation activity.

/'<ehcWbZ[^oZ[Yedl[hi_ed[\\_Y_[dYoThe fluorescent Histone Demethylase Assay
is designed to detect the formaldehyde
released from the reaction of lysine specificdemethylase 1 (LSD1, also known as KDM1)
with a methylated substrate. The recombinant histone H3K4me2 substrate used in
the assay mimics a native histone substrate,
generating results that more closely resemblein vivo conditions. As the LSD1 enzyme
demethylates the recombinant histone
substrate, formaldehyde is released as a byproduct. The Detection Reagent reacts witheach formaldehyde molecule to generate afluorescent signal equivalent to the overall
production of formaldehyde.

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CJ;("I\ZfdY`eXek?*B+d\)gifk\`eFigure 4: Comparison of different histone substrates
and their effect on LSD1 demethylase efficiency.
The positive control LSD1 enzyme from the Histone
Demethylase Assay was used to evaluate demethylase
activity using either a histone H3K4me2 peptide or the
kit’s recombinant histone H3K4me2 protein. One µg of
LSD1 was tested with either 70 µM H3K4me2 peptide
or with 13 µM recombinant histone H3K4me2 protein.
LSD1 was able to convert 73% of the recombinanthistone substrate into a formaldehyde by-product, yet
it was only able to convert 14% of the peptide substrate into a formaldehyde by-product, even though
there was 5-fold more peptide available than recombinant protein for the same amount of LSD1 enzyme.

HeLa Acid Extracts
Active Motif’s HeLa acid extracts are a reliable control for studying histone modifications. Extracts are available either untreated,or treated with chemicals known to affectepigenetic events, such as sodium butyrate,paclitaxel, etoposide and anacardic acid.

To learn more, or to see a complete listof available extracts, please visit us atwww.activemotif.com/acidextract.
www.activemotif.com

Recombinant Histone Proteins
Active Motif is the first company to offer
recombinant histones with acetylation andsite-specific mono-, di- and trimethylation.
These recombinant histones can be used ascontrols for histone antibodies, substrates
for histone modification enzymes, or to generate chromatin in vitro, using Active Motif’s
Chromatin Assembly Kit (Catalog No. 53500).
Recombinant methylated lysine residues are
created using a patented approach in which
an analog of methyl lysine is installed in thehistone via chemical alkylation. This enables
the site and degree of methylation to becarefully controlled. Each methylation reaction is over 99% complete and is verified
by high-resolution mass spectrometry. The
recombinant histones are also analyzed by
dot blot to confirm identity (Figure 5).
Acetylated histones are created with ourpatent pending technology that enables us
to acetylate the histone tail, without altering
the native peptide bonds. The ability of theacetylated and methylated histones to mimic
their native counterparts enables these substrates to yield more “natural” results thanhistone peptides.

Figure 5: Dot blot of recombinant histones.
One µg of unmodified, mono-, di- or trimethylated
recombinant proteins for H3K27, H3K36, H3K79 andH4K20 were spotted onto a PVDF membrane andprobed with Histone H3 dimethyl Lys79 pAb (Catalog
No. 39143) at a 1:1000 dilution. The dot blot confirms
the identity of the recombinant H3K79me2 protein.

For a complete list of our over 20 unique recombinant histones, please visit us at www.activemotif.com/recombhis.

Histone Modification ELISAs

The Histone Modification ELISA Kits provide
a sensitive method for detecting changes in
the level of specific histone modifications
from purified core histones, or histones
isolated by acid extraction. These easy-to-use
kits are sandwich ELISAs that utilize a capture

antibody against histone H3 and a detecting antibody specific to the modification of interest. An
HRP-conjugated secondary antibody and developing solutions provide a colorimetric readout inless than 3 hours (Figure 6).
Each kit includes validated modification
specific controls. The included methylated
recombinant histone proteins can be used
to generate a standard curve, enabling
quantification of the amount of site- anddegree-specific methylated histone in eachsample. The acid extracts provided in thephosphorylated ELISAs serve as a qualitative
control. The recombinant histones and acidextracts are also available separately (seeabove and on previous page).

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To better understand the effects of histone
modifications on chromatin remodeling andtranscriptional regulation, Active Motif has
developed over 10 different assays for important histone modification sites, such as lysine
methylation at K4, K9 and K27 or serine
phosphorylation at S10 and S28. These modification sites serve as key targets of histone
methyltransferase and histone demethylase
enzymes, or act as markers for mitosis.

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Figure 6: Histone H3 monomethyl Lys27 specificity.
Recombinant Histone H3, mono-, di- and trimethyl
Lys27 proteins were assayed from 15 ng - 1 µg per
well using the Histone H3 monomethyl Lys27 ELISA.
The results show the specificity of the assay for themonomethyl modification. The monomethyl protein is
included in the assay for sample quantification.

To see an up-to-date list of the more than 10 Histone Modification ELISAs available, please visit www.activemotif.com/hiselisa.

Chromatin Assembly
Design your own chromatin with Active
Motif’s Chromatin Assembly Kit. Using either
purified core histones, or Active Motif’s recombinant histones, combine histones with the kit

components and incubate with DNA to generateassembled chromatin that functions in a context
that closely resembles in vivo chromatin.

Product

FormatCatalog No.

Chromatin Assembly Kit

10 rxns

53500

The Chromatin Assembly Kit is an ATP-dependent
method that utilizes purified recombinant humanchromatin assembly complex ACF and histone
chaperone NAP-1 with core histones for in vitro
assembly of extended, regularly ordered, periodic
arrays of nucleosomes. For details, please visit
www.activemotif.com/chromassembly.

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www.activemotif.com

Histone Acetyltransferase (HAT) Activity
The Histone Acetyltransferase (HAT) Assay Kit
is a quick and sensitive method to determine
the activity of your own source of purified
histone acetyltransferases, or to screen forpotential inhibitors of HAT activity.
This fluorescent 96-well plate assay includes
N-terminal histone H3 and H4 substrate
peptides for screening HAT enzymes and apositive control p300 catalytic domain
protein to screen inhibitors. HATs will
catalyze the transfer of acetyl groups fromthe provided acetyl-CoA to generate anacetylated peptide and CoA-SH. After
stopping the reaction with stop solution, adeveloper is added that reacts with the freesulfhydryl groups on the CoA-SH to give afluorescent signal (Figure 7).
A standard curve can be generated witheither b-mercaptoethanol or acetyl-CoA inorder to relate the fluorescence of your HAT
to pmol/min/µg specific activity.

Product

FormatCatalog No.

HAT Assay Kit (Fluorescent)1 x 96 rxns

56100

5 µg

31205

Recombinant p300 protein, catalytic domain

Figure 7: HAT inhibitor effects on p300 activity.
The HAT activity of 50 ng p300 catalytic domain was
assayed using 50 µM acetyl-CoA and either 50 µM
Histone H3 or Histone H4 peptides. The addition of
15 µM anacardic acid or 25 µM garcinol inhibited the
HAT activity down to background levels. The background signal indicates the level of autoacetylation
present from the p300 acetyltransferase.

Histone Deacetylase (HDAC) Activity
cofactor to the assay.) Once the substrateis deacetylated, the lysine reacts with thedeveloping solution and releases either achromophore or a fluorophore, which is thenmeasured (Figure 8). A deacetylated assay
standard is provided in each kit to enablecalculation of HDAC activity in pmol/min/mg.
Additionally, the HDAC Assay Kits can beused to screen inhibitor compounds.

Product

FormatCatalog No.

HDAC Assay Kit (Fluorescent)1 x 96 rxns

56200

HDAC Assay Kit (Colorimetric)

1 x 96 rxns

56210

50000

Trichostatin A
Untreated

Fluorescence Intensity

To measure HDAC activity in your nuclear
extracts, immunoprecipitates, column fractions or purified proteins, Active Motif offers
your choice of fluorescent or colorimetricdetection. Both HDAC kits utilize a short
peptide substrate containing an acetylated
lysine residue that can be deacetylated byClass I, II and IV HDAC enzymes. (Class III &Sirtuin HDACs require the addition of NAD+

40000
30000
20000
10000
0

10

5

2.5

1.25

0.625

0.312

0.156

0

HeLa Nuclear Extract (µg)

Figure 8: Fluorescent HDAC assay results.
HeLa nuclear extracts were assayed for HDAC activity
in duplicate from 0 to 10 µg per well in the presence (copper line) or absence (purple line) of 1 µM
Trichostatin A inhibitor.

Histone Binding Assay to Identify Chromatin-modifying Proteins
Active Motif’s HiLite™ Binding Assay is aninnovative tool for identifying chromatinmodifying proteins that bind to the histone
tails of methylated H3K9 and H3K27. This
fast, homogeneous assay uses fluorescencepolarization (FP) to measure the affinity ofthe binding interactions between your protein of interest and specific histone methyla-tion states, which in turn enables fast andefficient inhibitor screening studies.

Product

FormatCatalog No.

HiLite™ Histone H3 Methyl-Lys9 / Lys27
Binding Assay

1 kit

57001

Each kit contains 8 fluorescently labeled
peptides that correspond to unmodified,mono-, di- and trimethylated histone H3lysine 9 and lysine 27 regions, as well as apositive control HP1 protein, assay buffer,calibration dye and five 96-well half area
black polystyrene plates. When the protein
of interest binds the modified peptide, it
slows the rotation of the peptide, causing
the amount of polarized light that is emitted to be greater than an unbound peptide.
This provides a quantitative measure of thehistone binding protein’s affinity for the
peptide’s histone modification.

To learn more about how this FP assay
works, visit www.activemotif.com/hilite.

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