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.