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HTRF Anti histone H3K4 Me2-EuK mAb conjugate, 500 Assay Points
This Eu-cryptate conjugate allows specific detection of the K4Me2 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
Product information
Overview
Revvity 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 H3K4Me2 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, such as JARID1A or 1C.
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
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.
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
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 H3K4 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 | |
|---|---|---|---|---|---|
| LSD1 | Demethylase | K4 me1 →me0 | H3(1-21)K4me1 | H3K4me0 | |
| LSD2 | Demethylase | K4 me2 →me0 | H3(1-21)K4me2 | H3K4me0 | |
| SET7/9 | Methyl transferase | K4 me0 →me1 | H3(1-21)K4me0 | H3K4me1 | |
| JARID1A | Demethylase | K4 me3 →me2 | H3(1-21)K4me3 | H3K4me2 | |
| JARID1C | Demethylase | K4 me3 →me2 | H3(1-21)K4me3 | H3K4me2 | |
| MLL1/WARD | Methyl transferase | K4 me0 →me2 | H3(1-50)K4me0 | H3K4me2 | |
| MLL1/WARD | Methyl transferase | K4 me0 →me3 | H3(1-50)K4me0 | H3K4me3 | |
| HDAC2 | Demethylase | K4 Ac →unmodified | H3(1-21)K4Ac | H3K4me0 |
Resources
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
Are you looking for technical documents for this product. We have housed them in a dedicated section., click on the links below to explore.
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