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HTRF Anti histone H3K9 Me1-EuK mAb conjugate, 500 Assay Points

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

For research use only. Not for use in diagnostic procedures.
Product Variants
Part number: 61KB1KAE
Unit Size: 500 Assay Points
List price: USD 1,304.00
Your price:
USD 1,304.00
USD 1,304.00 /each
Part number: 61KB1KAD
Unit Size: 10,000 Assay Points
List price: USD 7,973.00
Your price:
USD 0.00
USD 7,973.00 /each

Overview

Revvity 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.

Specifications

Assay Points
500
Assay Target Type
Fluorescent reagent
Assay Technology
HTRF
Brand
HTRF
Quantity
1
Therapeutic Area
Metabolism/Diabetes
Neuroscience
Oncology & Inflammation
Unit Size
500 Assay Points

Video gallery

How it works

Assay principle

Revvity’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).

HTRF Anti histone H3K9 Me1-EuK mAb conjugate 1.svg

 

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.

HTRF Anti histone H3K9 Me1-EuK mAb conjugate 2.svg

 

Assay details

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

Me2

V

 

V

 

 

Antibody specificity

Each Eu-cryptate labeled anti-histone antibody recognizes a single histone H3 lysine residue and methylation level. Revvity 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%

 

Assay validation

Validation of enzymatic assays

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

 

Resources

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Guide
HTRF solutions, guide to major applications

This guide provides you an overview of HTRF applications in several therapeutic areas.

SDS, COAs, Manuals and more Illuminator

SDS, COAs, Manuals and more

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