Anti histone H3K9 Me0-EuK mAb conjugate
Eu-cryptate conjugate for H3K9Me0 epigenetic mark detection
Anti histone H3K9 Me2-EuK mAb conjugate HTRF®
This Eu-cryptate conjugate allows specific detection of the K9Me2 motif on histone H3, as a result of methylation or demethylation enzymatic activity.
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Highly specific
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High sensitivity
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 H3K9Me2 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).
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 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 | |
| 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 | K9 | Ac →unmodified | H3(1-21)K9Ac | H3K9me0 | |
| HDAC2 | Deacetylase | K9 | Ac →unmodified | H3(1-21)K9Ac | H3K9me0 | |
| HDAC3 | Deacetylase | K9 | Ac →unmodified | H3(1-21)K9Ac | H3K9me0 | |
| HDAC6 | Deacetylase | K9 | Ac →unmodified | H3(1-21)K9Ac | H3K9me0 | |
| HDAC8 | Deacetylase | K9 | Ac →unmodified | H3(1-21)K9Ac | H3K9me0 | |
| SIRT1 | Deacetylase | K9 | Ac →unmodified | H3(1-21)K9Ac | H3K9me0 | |
| SIRT2 | Deacetylase | K9 | Ac →unmodified | H3(1-21)K9Ac | H3K9me0 | |
| SIRT3 | Deacetylase | K9 | Ac →unmodified | H3(1-21)K9Ac | H3K9me0 | |
| SIRT6 | Deacetylase | K9 | Ac →unmodified | H3(1-21)K9Ac | H3K9me0 | |
HTRF G9a Histone H3K9 di-methylation assay me0-- >me2
Assay protocol and validations - Technical Notes
HTRF G9a Histone H3K9 mono-methylation assay me1-->me2
Assay protocol and validations - Technical Notes
HTRF JMJD2C Histone H3K9 mono-demethylation assay me3-->me2
Assay protocol and validations - Technical Notes
Histone Demethylase JARID1A and JARID1C Enzymatic applications with HTRF® Technology
High-Throughput and Homogeneous assays - Posters
A comparison of detection technologies for the epigenetic enzyme JMJD2C histone H3 demethylase
HTRF, Lance & Alpha Lisa comparison - Posters
Setting-up HTRF technology for G9a & DNMT1 High-Throughput Screening
In collaboration with Universidad de Navarra - Posters
Build your own robust HTRF® assay using Cisbio's anti-histone cryptate labeled antibodies - Flyers
Manual Bioch. H3K9 Me2 / 61KB2KAD-61KB2KAE
61KB2KAD-61KB2KAE - Product Insert
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
Technical Data Sheet Bioch. H3K9 Me2 / 61KB2KAD
61KB2KAD Batch 05A - Batch Information
Technical Data Sheet Bioch. H3K9 Me2 / 61KB2KAD
61KB2KAD Batch 06A - Batch Information
Technical Data Sheet Bioch. H3K9 Me2 / 61KB2KAD
61KB2KAD Batch 07A - Batch Information
Technical Data Sheet Bioch. H3K9 Me2 / 61KB2KAD
61KB2KAD Batch 08A - Batch Information
Technical Data Sheet Bioch. H3K9 Me2 / 61KB2KAD
61KB2KAD Batch 09A - Batch Information
Technical Data Sheet Bioch. H3K9 Me2 / 61KB2KAD
61KB2KAD Batch 09RA - Batch Information
Safety Data Sheet (DEU) Bioch. H3K9 Me2 / 61KB2KAD
61KB2KAD - Safety Data Sheet
Safety Data Sheet (ELL) Bioch. H3K9 Me2 / 61KB2KAD
61KB2KAD - Safety Data Sheet
Safety Data Sheet (FRA-FR) Bioch. H3K9 Me2 / 61KB2KAD
61KB2KAD - Safety Data Sheet
Safety Data Sheet (ITA) Bioch. H3K9 Me2 / 61KB2KAD
61KB2KAD - Safety Data Sheet
Safety Data Sheet (SPA) Bioch. H3K9 Me2 / 61KB2KAD
61KB2KAD - Safety Data Sheet
Safety Data Sheet (ENG-GB) Bioch. H3K9 Me2 / 61KB2KAD
61KB2KAD - Safety Data Sheet
Safety Data Sheet (ENG-US) Bioch. H3K9 Me2 / 61KB2KAD
61KB2KAD - Safety Data Sheet
Technical Data Sheet Bioch. H3K9 Me2 / 61KB2KAE
61KB2KAE Batch 05A - Batch Information
Technical Data Sheet Bioch. H3K9 Me2 / 61KB2KAE
61KB2KAE Batch 08A - Batch Information
Technical Data Sheet Bioch. H3K9 Me2 / 61KB2KAE
61KB2KAE Batch 07A - Batch Information
Technical Data Sheet Bioch. H3K9 Me2 / 61KB2KAE
61KB2KAE Batch 09A - Batch Information
Safety Data Sheet (DEU) Bioch. H3K9 Me2 / 61KB2KAE
61KB2KAE - Safety Data Sheet
Safety Data Sheet (ELL) Bioch. H3K9 Me2 / 61KB2KAE
61KB2KAE - Safety Data Sheet
Safety Data Sheet (FRA-FR) Bioch. H3K9 Me2 / 61KB2KAE
61KB2KAE - Safety Data Sheet
Safety Data Sheet (ITA) Bioch. H3K9 Me2 / 61KB2KAE
61KB2KAE - Safety Data Sheet
Safety Data Sheet (SPA) Bioch. H3K9 Me2 / 61KB2KAE
61KB2KAE - Safety Data Sheet
Safety Data Sheet (ENG-GB) Bioch. H3K9 Me2 / 61KB2KAE
61KB2KAE - Safety Data Sheet
Safety Data Sheet (ENG-US) Bioch. H3K9 Me2 / 61KB2KAE
61KB2KAE - Safety Data Sheet
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