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Anti histone H3K27 Me3-EuK mAb conjugate

Anti histone H3K27 Me3-EuK mAb conjugate HTRF®

This Eu-cryptate conjugate allows specific detection of the K27Me3 motif on histone H3, as a result of methylation enzymatic activity.

Highly specific
High sensitivity

This Eu-cryptate conjugate allows specific detection of the K27Me3 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 H3K27Me3 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 EZH2.

Benefits

METHYLATION MARK SPECIFIC
STANDARDIZED ASSAY FORMAT
HTS AND PROFILING

Assay principle

Cisbios 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	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.

PeptideMAb 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%

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.

Enzyme	Type	Methylation level		Biotinylated substrate	Eu3+-Cryptate MAb	Application note
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

HTRF EZH2(Y641F) Histone H3K27 tri-methylation assay me0 -->me3

Assay protocol and validations - Technical Notes

Histone Demethylase JARID1A and JARID1C Enzymatic applications with HTRF® Technology

High-Throughput and Homogeneous assays - Posters

Product Insert Bioch. H3K27 Me3 / 61KC3KAD-61KC3KAE

61KC3KAD-61KC3KAE - Product Insert

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

Batch Information Bioch. H3K27 Me3 / 20231014

61KC3KAD Batch 007 - Batch Information

Safety Data Sheet (DEU) Bioch. H3K27 Me3 / 61KC3KAD

61KC3KAD - Safety Data Sheet

Safety Data Sheet (ELL) Bioch. H3K27 Me3 / 61KC3KAD

61KC3KAD - Safety Data Sheet

Safety Data Sheet (FRA-FR) Bioch. H3K27 Me3 / 61KC3KAD

61KC3KAD - Safety Data Sheet

Safety Data Sheet (ITA) Bioch. H3K27 Me3 / 61KC3KAD

61KC3KAD - Safety Data Sheet

Safety Data Sheet (SPA) Bioch. H3K27 Me3 / 61KC3KAD

61KC3KAD - Safety Data Sheet

Safety Data Sheet (ENG-GB) Bioch. H3K27 Me3 / 61KC3KAD

61KC3KAD - Safety Data Sheet

Safety Data Sheet (ENG-US) Bioch. H3K27 Me3 / 61KC3KAD

61KC3KAD - Safety Data Sheet

Batch Information Bioch. H3K27 Me3 / 20231014

61KC3KAE Batch 07C - Batch Information

Safety Data Sheet (DEU) Bioch. H3K27 Me3 / 61KC3KAE

61KC3KAE - Safety Data Sheet

Safety Data Sheet (ELL) Bioch. H3K27 Me3 / 61KC3KAE

61KC3KAE - Safety Data Sheet

Safety Data Sheet (FRA-FR) Bioch. H3K27 Me3 / 61KC3KAE

61KC3KAE - Safety Data Sheet

Safety Data Sheet (ITA) Bioch. H3K27 Me3 / 61KC3KAE

61KC3KAE - Safety Data Sheet

Safety Data Sheet (SPA) Bioch. H3K27 Me3 / 61KC3KAE

61KC3KAE - Safety Data Sheet

Safety Data Sheet (ENG-GB) Bioch. H3K27 Me3 / 61KC3KAE

61KC3KAE - Safety Data Sheet

Safety Data Sheet (ENG-US) Bioch. H3K27 Me3 / 61KC3KAE

61KC3KAE - Safety Data Sheet

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