This Eu-cryptate conjugate allows specific detection of the K9Me0 motif on histone H3, as a result of demethylation or deacetylation enzymatic activity.
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  • Highly specific
  • High sensitivity
This Eu-cryptate conjugate allows specific detection of the K9Me0 motif on histone H3, as a result of demethylation or deacetylation 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 H3K9Me0 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 DMTs such as JMJD1A, and HDACs 1, 3-8, as well as SIRT1-3 and 6.

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 level K4 K9 K27 K36
Me0 V V
Me1 V V V
Me2 V V V V
Me3 V 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 H3K9 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
JMJD1ADemethylaseK9me2 →me0H3(1-21)K9me2H3K9me0PDF
G9aMethyl transferaseK9me0 →me2H3(1-21)K9me0H3K9me2PDF
G9aMethyl transferaseK9me1 →me2H3(1-21)K9me1H3K9me2PDF
JMJD2CDemethylaseK9me3 →me2H3(1-21)K9me3H3K9me2PDF
HDAC1DeacetylaseK9Ac →unmodifiedH3(1-21)K9AcH3K9me0PDF
HDAC2DeacetylaseK9Ac →unmodifiedH3(1-21)K9AcH3K9me0PDF
HDAC3DeacetylaseK9Ac →unmodifiedH3(1-21)K9AcH3K9me0PDF
HDAC6DeacetylaseK9Ac →unmodifiedH3(1-21)K9AcH3K9me0PDF
HDAC8DeacetylaseK9Ac →unmodifiedH3(1-21)K9AcH3K9me0PDF
SIRT1DeacetylaseK9Ac →unmodifiedH3(1-21)K9AcH3K9me0PDF
SIRT2DeacetylaseK9Ac →unmodifiedH3(1-21)K9AcH3K9me0PDF
SIRT3DeacetylaseK9Ac →unmodifiedH3(1-21)K9AcH3K9me0PDF
SIRT6DeacetylaseK9Ac →unmodifiedH3(1-21)K9AcH3K9me0PDF
HTRF JMJD1A Histone H3K9 di-demethylation assay me2-->me0

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