This Eu-cryptate conjugate allows specific detection of the K27Me2 motif on histone H3, as a result of methylation or demethylation enzymatic activity.
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  • Highly specific
  • High sensitivity
This Eu-cryptate conjugate allows specific detection of the K27Me2 motif on histone H3, as a result of methylation or demethylation enzymatic activity.
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Overview

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. This monoclonal antibody Eu-cryptate conjugate is specific for the histone H3K27Me2 methylation mark. It can be used for the development of biochemical assays with the highest levels of specificity and sensitivity, for the screening and study of both HMTs and HDMs, such as G9a.

Assay principle

Cisbio’s HTRF-based reagents give researchers the flexibility to create highly specific, custom histone modification assays for enzymes such as HMTs, DMTs or HDACs. All these assays use a biotinylated histone H3 peptide substrate, a Eu-cryptate labeled detection antibody and XL665-conjugated Streptavidin. Assay specificity is derived from the combination of the chosen Eu-cryptate labeled anti-histone antibody together with the biotinylated peptide substrate. HTRF signal is proportional to the concentration of methyl-specific peptide (e.g. unmethylated, mono-, di- or tri-methylated peptide).

Assay protocol

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 Eu-labeled anti-methylated histone H3 antibody and SA-XL665 are added. The HTRF signal is detected following a brief room temperature incubation, and is stable for multiple readouts.

Anti-histone H3 Eu-cryptate conjugates

Methylation levelK4K9K27K36
Me0VV

Me1VV
VV
Me2VVVV
Me3V
V

Antibody Specificity

Each Eu-cryptate labeled anti-histone antibody recognizes a single histone H3 lysine residue and methylation level. Cisbio has performed cross-reactivity studies against alternative peptides and methylation levels for each antibody to ensure specificity.
PeptideMAb
H3K4H3K9H3K27H3K36
me0me1me2me3me0me1me2me1me2me3me1me2
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) K4Ac0%-----------
H3(1-21) K9Ac----0.1%-------
H3(1-21) K4me10%100%0.6%0.5%--0%--0%--
H3(1-21) K4me20%15%100%7.4%--0%--0%--
H3(1-21) K4me30%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%

Validation of enzymatic assays

Cisbio has developed a number of HTRF assays for HMT/HDM/HDAC enzymes using the antibody conjugate epigenetic panel. The table below recaps the assay set up for H3K27 position, and provides in each case a technical note including materials and methods as well as the results obtained.
EnzymeTypeMethylation levelBiotinylated substrateEu3+-Cryptate MAbApplication note
G9aMethyl transferaseK27me0 →me1H3(21-44)K27me0H3K27me1PDF
G9aMethyl transferaseK27me0 →me2H3(21-44)K27me0H3K27me2PDF
EZH2(Y641F)Methyl transferaseK27me0 →me3H3(1-50)K27me0H3K27me3PDF
HTRF G9a Histone H3K27 di-methylation assay me0 -->me2

Assay protocol and validations - Technical Notes

EPIgeneous Toolbox Reagents

Build your own robust HTRF® assay using Cisbio’s anti-histone cryptate labeled antibodies - Flyers

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

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