EPIgeneous H3K27Me3 cellular kit

This cell-based assay format enables the detection of the H3K27Me3 motif, as a result of histone H3 methylation.
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  • Cell-based
  • High sensitivity
This cell-based assay format enables the detection of the H3K27Me3 motif, as a result of histone H3 methylation.
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Overview

This cellular assay has been developed with optimized reagents and protocols for the direct detection of endogenous levels of H3K27Me3.

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, for the study of important epigenetic targets, such as EZH1 and 2.

The H3K36Me2 assay can be used for adherent or suspension cells, primary or secondary screening, and inhibitor studies.

Assay principle

The trimethylation of Lysine 27 on histone H3 is detected in a sandwich assay format using 2 different specific antibodies, one labeled with Eu-Cryptate (donor) and the second with d2 (acceptor). When the dyes are in close proximity, the excitation of the donor with a light source triggers a Fluorescence Resonance Energy Transfer (FRET), which in turn fluoresces at a specific wavelength (665 nm). One conjugate binds to Histone H3 and the other binds to the K27Me3 mark. The specific signal modulates positively in proportion to trimethylation on Lysine 27.

Two-plate assay protocol

Cells are plated (stimulated) and lysed in the same culture plate and then transferred to the assay plate for the detection of H3K27Me3 by HTRF reagents. This protocol enables the cells’ viability and confluence to be monitored.

One-plate assay protocol

Detection of H3K27Me3 with HTRF reagents is performed in a single plate used for plating, stimulation and detection. No washing steps are required. This protocol, HTS designed, allows miniaturization while maintaining HTRF quality.

Detection of H3K27Me3 over time on the 2 cell lines

HTRF detection of H3K27Me3 mark with a two plate protocol in two cell lines: SU-DHL-6 (Heterozygous mutation of EZH2 (Y461N) affecting substrate selectivity leading to high level of H3K27Me3), and OCI-LY-19 (weak level of H3K27me3, mostly accumulates H3K27me2).

Cells were seeded at various densities in a 96-well plate and incubated for several different times before lysis and detection in a 384sv plate.

The H3K27Me3 modulation observed between the two cell lines is about 5 up to 6 fold at a fixed optimal concentration. Optimization of cell densities is mandatory in order to establish the best conditions for the incubation time sought.

For example, according to the data, if 72h incubation is required, the best cell concentration would be between 10 000 and 20 000 cells per well.

In fact, for this incubation time a hook effect is appears for the 30 000 cells per well condition.

Antibody Specificity

SU-DHL-6 were seeded at a density of 30 000 cells per well in 96-well plate for a 24h* incubation.

Serial dilutions of Histone H3 derived peptides (from 10µM down to 13.7nM) with various epigenetic methyl marks were added to the detection well before the addition of HTRF detection conjugates.

Only peptides with H3K27Me3 competed with the system, with an IC50 value of 70 nM showing the specificity of the kit.

*Cell lysates were transfered to a 384-well plate.

Inhibition curves (GSK126 compound)

​HTRF detection of H3K27Me3 mark with a two plate protocol. SU-DHL-6 and OCI-LY-19 cells were seeded at 20 000cells/w in 96-well plate. Cells were treated for 72h with selective EZH2 inhibitor GSK126 (from 10µM to 13.7nM diluted in medium). GSK 126 showed an IC50 value of 70nM on SUDHL6 and 10nM on OCI-LY-19 cell line.

Using EPIgeneous Total H3 kit, effect of GSK 126 on total H3 was monitored. Except for the 10µM concentration, no effect of GSK 126 on Total H3 was observed.

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