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Anti histone H3K9 Me1-EuK mAb conjugate HTRF®

This Eu-cryptate conjugate allows specific detection of the K9Me1 motif on histone H3, as a result of methylation or demethylation enzymatic activity.

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  • Highly specific Highly specific
  • High sensitivity High sensitivity

This Eu-cryptate conjugate allows specific detection of the K9Me1 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) and demethylase (HDM) activity assays. This monoclonal antibody Eu-cryptate conjugate is specific for the histone H3K9Me1 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.

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

Me1VVVV
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 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 G9a Histone H3K9 mono-methylation assay me0-->me1

Assay protocol and validations - Technical Notes

Product Insert Bioch. H3K9 Me1 / 61KB1KAD-61KB1KAE

61KB1KAD-61KB1KAE - Product Insert

HTRF Product Catalog 2020 July update

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

HTRF G9a Histone H3K9 mono-methylation assay me1-->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

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

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