Histone Peptide Array

NEW: Screen Protein Domains for Histone Binding Partners
Active Motif was the first company to introduce an array capable of analyzing antibody and protein interactions
with histone post-translational modifications (PTMs). Our unique MODified™ Histone Peptide array offers the ability
to screen 384 different modification combinations to evaluate cross-reactivity between individual modifications aswell as investigating the effects of adjacent modifications on binding affinity. Active Motif has expanded our product offering with our new MODified™ Protein Domain Binding Kit. This assay is designed for researchers interested inscreening their protein domains (e.g. bromodomains, chromodomains, etc.) for reactivity with specific histone PTMs.

What are protein domains?
The epigenetic information that exists inthe form of post-translational modifications on histone tails is generated,interpreted and edited by proteins thatare coined “writers”, “readers” and “erasers”. There are several classes of protein
domains that influence gene regulationand chromatin remodeling by interactingwith specific histone PTMs. See Table 1for a list of some common chromatin
remodeling protein domains.
Our current understanding of these
protein domains has been limited by thelack of appropriate tools to fully interrogate their binding specificity. Active
Motif’s MODified™ Histone Peptide Arrayprovides a solution to this problem as
it enables researchers to screen a large
panel of histone modifications in a singleexperiment. The results provide informa-tion on protein domain interactions withspecific histone modifications, and also
reveals the influence that adjacent modifications may play on protein binding.
How does it work?
Active Motif’s MODified Histone
Peptide Array contains modifications
to study histone acetylation, methylation, phosphorylation or citrullination
on histones H2A, H2B, H3 and H4. Eachpeptide is spotted in duplicate on thearray to analyze the reproducibility ofthe binding events.
The MODified Protein Domain Binding
Kit was designed to be used in conjunction with the peptide arrays. The simple
assay works like a Western blot. The
array is first incubated in blocking buffer.
Then, the His-tagged protein domainof interest is added to the array anddetected via an anti-His tag antibody
and an HRP-conjugated secondary antibody. ECL detection is used to produce asignal. An image of the array is captured
using a luminescent imaging system.
Free Array Analyze Software
Active Motif offers free Array Analyze
software that can be downloaded fromour website to analyze the intensity ofthe spots. Information about binding
partners can be interrogated through thesoftware program to help identify thehistone modifications associated withbinding of the protein domain (Figure 1).
We have recently updated our Array
Analyze software package to include
new features that make the softwareeasier to use and simplify the way theresults are displayed. Download a copyof our new software program today atwww.activemotif.com/modified.

DomainBromo domain

Binding Site
Acetylated lysine residues onhistones or other proteins

Function
Regulates chromatin structure and gene expression as part ofhistone acetyltransferases or chromatin remodeling factors

ExamplesTAFII250, PCAF, GCN5

Chromo domain

Methylated lysine residues onHistone H3

Associated with the assembly of protein complexes onchromatinHP1b, MPP8, CHD1

Tudor domain

Methylated lysine or arginine
residues on Histone H3 and H4

It may be involved in RNA binding, DNA damage response andchromatin modificationJMJD2A, 53BP1, SMN

MBT domain

Methylated lysine residues onHistone H3 and H4

The exact function is unknown, but MBT often appears as repeats inproteins associated with transcriptional repression

L(3)MBTL, CGI-72

Table 1: Examples of some common protein domain classes and their associated binding specificity and function.

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Histone Peptide Array
Figure 1: Analysis of the JMJD2A protein
domain using the MODified Protein Domain
Binding Kit and the Modified Histone Array.
The His-tagged Tudor domain protein,
JMJD2A, was used at a 50 nM concentration
with the MODified Protein Domain Binding
Kit and the MODified Histone Peptide Array.
An image of the array was captured using achemiluminescent imaging system.
Top: JMJD2A array image in which the grid
overlay on the left half demonstrates thealignment performed by the Array Analyze
software program for spot identification.

Binding Specificity of JMJD2A

Average Intensity

1
0.8
0.6

Addition of modifications at R26
have little effect on binding at K27me3
Single K27me3
modification

0.4

Addition of S28phinhibits binding

0.2H4un
H4 mo
K2 d0m
H4 e3K16aHH
4H4 K 4 K c161K12 ac 2aac K2 c
K16 0mac e3
K20H3 me3un
H3 modK2
H3 7meR2 3
H3 6meR2 2s6m
H3R2 H3 R e2a
H3 6me 26CR2 2s K itr
6m 2
H3 e2a 7me
R2 K2 36C 7m
H3ite
R2 H3 K r K2 376
H3 me2 27m me3
R2 s K e3
6m 27 S28
e2 me phaK 3S27m 28
e3 ph
S28ph0

MODified™ Array Peptide Identity

MODified Array Labeling Kit
In addition to the new Protein Domain
Binding Kit, the MODified Histone
Peptide Arrays can also be used toscreen antibodies for cross-reactivity.
The MODified™ Array Labeling Kit
provides all the reagents needed for theblocking, washing and chemiluminescentdetection of the array. HRP-conjugatedsecondary antibodies for use with either
rabbit or mouse primary antibodies, anda positive control antibody that recognizes the c-Myc control spot printed
on the peptide array are also included.
The c-Myc control antibody can becombined with the antibody you arescreening to assist with spot identification within the Array Analyze software.

2Bottom: Graphical analysis of selected datafrom the Array Analyze software program which
was used to calculate the spot intensity ofbinding events for the JMJD2A protein domain.
The average intensity was calculated for each
duplicate on the left and right side of the arrayand graphed for the peptides listed.
The data shows the specificity of the JMJD2A
domain for Histone H4K20me3 and H3K27me3(green arrow). The presence of additional modifications within the same peptide had varying
influences on spot intensity. There was relatively little change when the H3K27me3 modification was present together with modificationsat R26 (copper arrows). But, when the S28ph
modification was adjacent to K27me3, bindingof the JMJD2A protein domain was inhibited
greatly (red arrows).

Active Motif offers a broad selection of recombinant proteins,
including Bromodomains, HATs, HDACs, HMTs and HDMs, which can beused to study functional changes and binding specificity on theMODified Histone Peptide Array. To see a full list of available proteins,please visit us at www.activemotif.com/proteins.

ProductFormat

Catalog No.

MODified™ Histone Peptide Array

1 array
5 arrays

13001
13005

MODified™ Protein Domain Binding Kit

5 rxns13007

MODified™ Array Labeling Kit

5 rxns13006

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