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Anti histone H3K9 Me0-EuK mAb conjugate

Anti histone H3K9 Me0-EuK mAb conjugate HTRF®

This Eu-cryptate conjugate allows specific detection of the K9Me0 motif on histone H3, as a result of demethylation or deacetylation enzymatic activity.

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.

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
PeptideMAb	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 H3K9 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
JMJD1A	Demethylase	K9	me2 →me0	H3(1-21)K9me2	H3K9me0	PDF
G9a	Methyl transferase	K9	me0 →me2	H3(1-21)K9me0	H3K9me2	PDF
G9a	Methyl transferase	K9	me1 →me2	H3(1-21)K9me1	H3K9me2	PDF
JMJD2C	Demethylase	K9	me3 →me2	H3(1-21)K9me3	H3K9me2	PDF
HDAC1	Deacetylase	K9	Ac →unmodified	H3(1-21)K9Ac	H3K9me0	PDF
HDAC2	Deacetylase	K9	Ac →unmodified	H3(1-21)K9Ac	H3K9me0	PDF
HDAC3	Deacetylase	K9	Ac →unmodified	H3(1-21)K9Ac	H3K9me0	PDF
HDAC6	Deacetylase	K9	Ac →unmodified	H3(1-21)K9Ac	H3K9me0	PDF
HDAC8	Deacetylase	K9	Ac →unmodified	H3(1-21)K9Ac	H3K9me0	PDF
SIRT1	Deacetylase	K9	Ac →unmodified	H3(1-21)K9Ac	H3K9me0	PDF
SIRT2	Deacetylase	K9	Ac →unmodified	H3(1-21)K9Ac	H3K9me0	PDF
SIRT3	Deacetylase	K9	Ac →unmodified	H3(1-21)K9Ac	H3K9me0	PDF
SIRT6	Deacetylase	K9	Ac →unmodified	H3(1-21)K9Ac	H3K9me0	PDF

HTRF JMJD1A Histone H3K9 di-demethylation assay me2-->me0