EPIgeneous H3K4Me2 cellular kit
- High sensitivity
This cellular assay has been developed with optimized reagents and protocols for the direct detection of endogenous levels of H3K4Me2.
This HTRF® format uses a very specific antibody pair combined with a highly efficient nucleus extraction which enables the assay to be used with a variety of cell backgrounds, and for the study of important methyltransferases and demethylases targets, such as JARID1 and MLL1.
The H3K27Me3 assay can be used for adherent or suspension cells, primary or secondary screening, and inhibitor studies.
HeLa cells were seeded at a density of 15,000 cells per well in a 96-well plate for a 24 h incubation.
Serial dilutions of Histone H3 derived peptides (from 10 µM down to a minimum of 1 nM) with various epigenetic methyl marks were added to the detection well before the addition of HTRF detection conjugates.
H3K4Me1 peptide competed with low affinity (IC50 = 1.08 µM), while the H3K4Me2 peptide competed with high affinity with the system leading to an IC50 value of 26.7 nM showing the specificity of the kit.
HeLa cells were seeded at 12,500cells per well in a 96-well plate, then treated overnight with TSA, NaB and Apicidine, 3 well known HDAC inhibitors that induce the di-methylation of H3K4 (H3K4Me2) and a decrease of H3K9Me2.
After lysis, lysates were transfered to a 384-sv plate for detection using a two-plate assay protocol.
EPIgeneous cellular assays for measuring epigenetic modifications - Flyers
EPIgeneous Cell Based Assay Lysis Buffer Guide - Technical Notes