Epigenetic toolbox reagents

Validated antibodies for high specificity screening of histone modifications

Cisbio offers a line of ready to use HTRF® toolbox reagents for the construction of Histone H3 methyltransferase (HMT), demethylase (HDM) and deacetylase (HDAC) activity assays. Create your own homogeneous, miniaturized HTRF® assay using one of our europium-cryptate labeled antibodies or implement one of our validated assays to improve the throughput and reliability of your epigenetic screening. Each anti-histone HTRF® antibody is specific for a single histone H3 lysine residue and methylation mark enabling development of assays with the highest levels of specificity and sensitivity. Don’t have the time or resources to do the work yourself? Let the Cisbio HTRF® service team move your epigenetic research forward through our custom labeling and full epigenetic assay development services.

Antibody Selection

Cisbio offers an array of Eu3+- Cryptate labeled anti-histone H3 antibodies, each specific for a single H3 lysine residue and methylation state. These antibodies can be used in conjunction with a biotinylated peptide substrate and XL-665 conjugated Streptavidin to create a custom, high specificity HMT, HDM or HDAC assays. Cisbio has developed and validated a number of HTRF assays for HMT/HDM/HDAC enzymes using our epigenetic toolbox antibodies. Please refer to the product and assay tables below to identify HTRF antibodies of interest. Don’t see an antibody to the histone residue you need or a specific methylation level ? Cisbio offers a full range of custom labeling and assay development services.

Methylation level K4 K9 K27 K36
Me0    
Me1  
Me2
Me3    

 

Enzyme Type Methylation level Biotinylated substrate Eu3+-Cryptate MAb Application note
LSD1 Demethylase K4 me1 → me0 H3(1-21)K4me1 H3K4me0 PDF
LSD1 Demethylase K4 me2 → me0 H3(1-21)K4me2 H3K4me0  
SET7/9 Methyl transferase K4 me0 → me1 H3(1-21)K4me0 H3K4me1 PDF
JARID1A Demethylase K4 me3 → me2 H3(1-21)K4me3 H3K4me2 PDF
JARID1C Demethylase K4 me3 → me2 H3(1-21)K4me3 H3K4me2 PDF
MLL1/WARD Methyl transferase K4 me0 → me2 H3(1-50)K4me0 H3K4me2 PDF
MLL1/WARD Methyl transferase K4 me0 → me3 H3(1-50)K4me0 H3K4me3 PDF
JMJD1A Demethylase K9 me2 → me0 H3(1-21)K9 me2 H3K9me0 PDF
G9a Methyl transferase K9 me0 → me2 H3(1-21)K9me0 H3K9me2 PDF
G9a Methyl transferase K9 me1 → me2 H3(1-21)K9me1 H3K9me2 PDF
JMJD2C Demethylase K9 me3 → me2 H3(1-21)K9me3 H3K9me2 PDF
G9a Methyl transferase K27 me0 → me1 H3(21-44)K27me0 H3K27me1 PDF
G9a Methyl transferase K27 me0 → me2 H3(21-44)K27me0 H3K27me2 PDF
EZH2(Y641F) Methyl transferase K27 me0 → me3 H3(1-50)K27me0 H3K27me3 PDF
JHDM1A Demethylase K36 me2 → me1 H3(21-44)K36me2 H3K36me1 PDF
JMJD2A Demethylase K36 me3 → me2 H3(21-44)K36me3 H3K36me2 PDF
HDAC1 Deacetylase K9 Ac → unmodified H3(1-21)K9Ac H3K9me0 PDF
HDAC2 Deacetylase K4 Ac → unmodified H3(1-21)K4Ac H3K4me0 PDF
HDAC2 Deacetylase K9 Ac → unmodified H3(1-21)K9Ac H3K9me0 PDF
HDAC3 Deacetylase K9 Ac → unmodified H3(1-21)K9Ac H3K9me0 PDF
HDAC6 Deacetylase K9 Ac → unmodified H3(1-21)K9Ac H3K9me0 PDF
HDAC8 Deacetylase K9 Ac → unmodified H3(1-21)K9Ac H3K9me0 PDF
SIRT1 Deacetylase K9 Ac → unmodified H3(1-21)K9Ac H3K9me0 PDF
SIRT2 Deacetylase K9 Ac → unmodified H3(1-21)K9Ac H3K9me0 PDF
SIRT3 Deacetylase K9 Ac → unmodified H3(1-21)K9Ac H3K9me0 PDF
SIRT6 Deacetylase K9 Ac → unmodified H3(1-21)K9Ac H3K9me0 PDF

Assay validation

Assay Principle

Cisbio’s line of HTRF-based reagents gives researchers the flexibility to create highly specific, custom histone modification assays. Typical HTRF methyltransferase (HMT), demethylase (HDM) and deacetylase (HDAC) assays use a biotinylated histone H3 peptide substrate, a Eu3+-cryptate labeled detection antibody and XL665-conjugated Streptavidin (SA-XL665). Assay specificity is derived from the combination of the chosen Eu3+-cryptate labeled anti-histone antibody as well as the biotinylated peptide substrate. HTRF signal is proportional to the concentration of methyl-specific peptide (e.g. unmethylated, mono-, di- or tri-methylated peptide).

Two step assays are performed in a single well, require no wash steps and can easily be miniaturized to a 384-well format. The enzymatic reaction containing the biotinylated peptide, enzyme, co-factors and compounds is followed by the detection step in which Eu3+-labeled anti-methylated histone H3 antibody and SA-XL665 are added. HTRF signal is detected following brief room temperature incubation, and is stable for multiple readouts.

HTRF reagents needed for histone modification assays

From Cisbio (see Part#, insert, & MSDS section)

  • Anti-histone antibody cryptate conjugate specific for a given methylation mark
  • XL665-conjugated streptavidin
  • Detection buffer

Positive control biotinylated peptides are available from Anaspec for each toolbox antibody (see antibody Package Inserts for Anaspec related references)

Antibody Specifity

Each Eu3+-cryptate labeled anti-histone antibody recognizes a single histone H3 lysine residue and methylation level. Cisbio performs cross-reactivity studies against alternate peptides and methylation levels for each antibody to ensure specificity. An example table of cross-reactivity is shown below.

Peptide\MAb H3K4 H3K9 H3K27 H3K36
me0 me1 me2 me3 me0 me1 me2 me1 me2 me3 me1 me2
H3(1-21) 100% 1.3% 0% 0% 100% 0.7% 0% - - 0% - -
H3(21-44) 0% - - 0% - - 0% 0% 0% 0% 4.4% 0%
H3(1-21) K4Ac 0% - - - - - - - - - - -
H3(1-21) K9Ac - - - - 0.1% - - - - - - -
H3(1-21) K4me1 0% 100% 0.6% 0.5% - - 0% - - 0% - -
H3(1-21) K4me2 0% 15% 100% 7.4% - - 0% - - 0% - -
H3(1-21) K4me3 0% 0.4% 0% 100% - - 0% - - 0% - -
H3(1-21) K9me1 - - - 0% 0.4% 100% 0.1% - - 0% - -
H3(1-21) K9me2 - - - 0% 0.3% 0% 100% - - 0% - -
H3(1-21) K9me3 - - - 0.2% 0.1% 0% 0.1% - - 0.2% - -
H3(21-44) K27me1 - - - 0% - - 0.1% 100% 1.5% 0.2% - 0%
H3(21-44) K27me2 - - - 0.2% - - 0% 1.4% 100% 3.3% - 0.1%
H3(21-44) K27me3 - - - 0% - - 0.1% 0% 0% 100% - 0%
H3(21-44) K36me1 - - - 0% - - 0.1% - - 0% 100% 0.7%
H3(21-44) K36me2 - - - 0% - - 0.1% - - 0.1% 0.60% 100%
H3(21-44) K36me3 - - - 0.1% - - 0.1% - - 0.4% 0% 0%
Peptide\MAb H3K4me0 H3K4me1 H3K4me2 H3K4me3 H3K9me0 H3K9me1 H3K9me2 H3K27me1 H3K27me2 H3K27me3 H3K36me1 H3K36me2
H3(1-21) 100% 1.3% 0% 0% 100% 0.7% 0% - - 0% - -
H3(21-44) 0% - - 0% - - 0% 0% 0% 0% 4.4% 0%
H3(1-21) K4Ac 0% - - - - - - - - - - -
H3(1-21) K9Ac - - - - 0.1% - - - - - - -
H3(1-21) K4me1 0% 100% 0.6% 0.5% - - 0% - - 0% - -
H3(1-21) K4me2 0% 15% 100% 7.4% - - 0% - - 0% - -
H3(1-21) K4me3 0% 0.4% 0% 100% - - 0% - - 0% - -
H3(1-21) K9me1 - - - 0% 0.4% 100% 0.1% - - 0% - -
H3(1-21) K9me2 - - - 0% 0.3% 0% 100% - - 0% - -
H3(1-21) K9me3 - - - 0.2% 0.1% 0% 0.1% - - 0.2% - -
H3(21-44) K27me1 - - - 0% - - 0.1% 100% 1.5% 0.2% - 0%
H3(21-44) K27me2 - - - 0.2% - - 0% 1.4% 100% 3.3% - 0.1%
H3(21-44) K27me3 - - - 0% - - 0.1% 0% 0% 100% - 0%
H3(21-44) K36me1 - - - 0% - - 0.1% - - 0% 100% 0.7%
H3(21-44) K36me2 - - - 0% - - 0.1% - - 0.1% 0.60% 100%
H3(21-44) K36me3 - - - 0.1% - - 0.1% - - 0.4% 0% 0%

Our assays have been validated using material from the following suppliers:

  • Enzymes from BPS Bioscience and from Cayman chemical
  • Peptidic substrates from Anaspec
  • Co-factors from Sigma

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Mab Anti histone H3K4 Me0-Eu(K) - 10,000 tests61KA0KAD
Mab Anti histone H3K4 Me0-Eu(K) - 500 tests61KA0KAE
Mab Anti histone H3K4 Me1-Eu(K) - 10,000 tests61KA1KAD
Mab Anti histone H3K4 Me1-Eu(K) - 500 tests61KA1KAE
Mab Anti histone H3K4 Me2-Eu(K) - 10,000 tests61KA2KAD
Mab Anti histone H3K4 Me2-Eu(K) - 500 tests61KA2KAE
Mab Anti histone H3K4 Me3-Eu(K) - 10,000 tests61KA3KAD
Mab Anti histone H3K4 Me3-Eu(K) - 500 tests61KA3KAE
Mab Anti histone H3K9 Me0-Eu(K) - 10,000 tests61KB0KAD
Mab Anti histone H3K9 Me0-Eu(K) - 500 tests61KB0KAE
Mab Anti histone H3K9 Me1-Eu(K) - 10,000 tests61KB1KAD
Mab Anti histone H3K9 Me1-Eu(K) - 500 tests61KB1KAE
Mab Anti histone H3K9 Me1-Eu(K) - 1mg61KB1KAH
-
Mab Anti histone H3K9 Me2-Eu(K) - 10,000 tests61KB2KAD
Mab Anti histone H3K9 Me2-Eu(K) - 500 tests61KB2KAE
Mab Anti histone H3K27 Me1-Eu(K) - 10,000 tests61KC1KAD
Mab Anti histone H3K27 Me1-Eu(K) - 500 tests61KC1KAE
Mab Anti histone H3K27 Me2-Eu(K) - 10,000 tests61KC2KAD
Mab Anti histone H3K27 Me2-Eu(K) - 500 tests61KC2KAE
Mab Anti histone H3K27 Me3-Eu(K) - 10,000 tests61KC3KAD
Mab Anti histone H3K27 Me3-Eu(K) - 500 tests61KC3KAE
Mab Anti histone H3K36 Me1-Eu(K) - 10,000 tests61KD1KAD
Mab Anti histone H3K36 Me1-Eu(K) - 500 tests61KD1KAE
Mab Anti histone H3K36 Me2-Eu(K) - 10,000 tests61KD2KAD
Mab Anti histone H3K36 Me2-Eu(K) - 500 tests61KD2KAE

Companion products

DescriptionCat. noProduct insertMSDS
Streptavidin-d2 - 5,000 tests610SADLA
Streptavidin-d2 - 20,000 tests610SADLB
Streptavidin-d2 - 1,000 tests610SADLF
-
Streptavidin-XL665 - 5,000 tests610SAXLA
Streptavidin-XL665 - 20,000 tests610SAXLB
Streptavidin-XL665 - 1,000 tests610SAXLF
-
Streptavidin-XLent! - 5,000 tests611SAXLA
Streptavidin-XLent! - 20,000 tests611SAXLB
HTRF detection buffer - 40 ml62SDBRDD

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