This Eu-cryptate conjugate allows specific detection of the K4Me3 motif on histone H3, as a result of methylation enzymatic activity.

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  • High sensitivity High sensitivity

This Eu-cryptate conjugate allows specific detection of the K4Me3 motif on histone H3, as a result of methylation 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 H3K4Me3 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 HMTs, such as MLL1.

Benefits

  • METHYLATION MARK SPECIFIC
  • STANDARDIZED ASSAY FORMAT
  • HTS AND PROFILING

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).
EPIgeneous histone modification assay principle

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.
EPIgeneous histone modification assay principle

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.
PeptideMAbH3K4H3K9H3K27H3K36
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 H3K4 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
LSD1DemethylaseK4me1 →me0H3(1-21)K4me1H3K4me0PDF
LSD1DemethylaseK4me2 →me0H3(1-21)K4me2H3K4me0
SET7/9Methyl transferaseK4me0 →me1H3(1-21)K4me0H3K4me1PDF
JARID1ADemethylaseK4me3 →me2H3(1-21)K4me3H3K4me2PDF
JARID1CDemethylaseK4me3 →me2H3(1-21)K4me3H3K4me2PDF
MLL1/WARDMethyl transferaseK4me0 →me2H3(1-50)K4me0H3K4me2PDF
MLL1/WARDMethyl transferaseK4me0 →me3H3(1-50)K4me0H3K4me3PDF
HDAC2DeacetylaseK4Ac →unmodifiedH3(1-21)K4AcH3K4me0PDF
HTRF MLL1/WARD Histone H3K4 tri-methylation assay me0-->me3

Assay protocol and validations - Technical Notes

EPIgeneous Toolbox Reagents

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Cisbio Product Catalog 2019

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