EPIgeneous™ H3K36Me2 Cellular Assay Kit

H3K36Me2 Cellular assay for studying methyltransferases and demethylases

This cellular assay has been developed with optimized reagents and protocols for the direct detection of endogenous levels of H3K36Me2. HTRF® is a flexible assay format enabling you to use an assay for a variety of applications, without having to compromise on sensitivity. The H3K36Me2 assay can be used for adherent, suspension cells, primary or secondary screening, and inhibitor studies of both methyltransferases and demethylases.

Assay principle

This assay is designed for the simple, rapid and direct detection of endogenous levels of H3K36Me2 mark in cells. The dimethylation of Lysine 36 on histone H3 is detected in a sandwich assay format using 2 different specific antibodies, one labelled with Eu3+-Cryptate (donor) and the second with d2 (acceptor). When the dyes are in close proximity, the excitation of the donor with a light source (laser or flash lamp) triggers a Fluorescence Resonance Energy Transfer (FRET) towards the acceptor, which in turn fluoresces at a specific wavelength (665 nm). One conjugate binds to Histone H3 and the other binds to H3K36Me2, thereby generating FRET. The specific signal modulates positively in proportion to dimethylation on Lysin 36.

The assays can be run under a two-plate protocol. They can also be further streamlined to a one-step assay.

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 H3K36Me2 by HTRF reagents. This protocol enables the cells’ viability and confluence to be monitored.

One-plate assay protocol

Detection of H3K36Me2 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.

Product performance

Detection of H3K36Me2

HTRF detection of H3K36Me2 mark with two plate protocol and HeLa cells. Cells were seeded at various densities in a 96-well plate and incubated for several different times before lysis. Panel A had a lysis step made without discarding the medium with the appropriate buffer. Panel B had a lysis step carried out when the medium was discarded. Detection of H3K36Me2 was performed in a 384-well white plate on a BMG Pherastar FS Flash lamp reader. Optimization of cell densities is required in order to establish the best condition suited for the incubation time desired. For example, whatever the incubation time, a hook effect occurred for the 30 000 cells per well concentration. A protocol on adherent cells, with the medium discarded prior to the lysis step, can enhance the assay's sensitivity.

Detection of H3K36Me2 with 2 different cell lines

HTRF detection of H3K36Me2 mark with the two plate protocol in two cell lines: HeLa and U2OS. Cells were seeded at various densities in a 96-well plate and incubated for 24h before lysis without medium. Detection of H3K36Me2 was performed in a 384w white plate on a BMG Pherastar FS Flash lamp reader. For both cell lines, a hook effect was observed for the 30,000 cells per well concentration. For the same cellular concentration, U2OS showed an increase level of dimethylation on H3K36.

Antibody Specificity

HeLa were seeded at a density of 12 000 cells per well in a 96-well plate and lysed after a 4 hour incubation. One concentration (10µM) of each Histone H3 derived peptide, bearing various epigenetic methyl marks, was added to the detection well before the addition of HTRF detection conjugates. As H3K36Me1 and H3K36Me2 peptides are the only peptides to compete with the antibodies, testing was carried out with serial dilutions in order to estimate the IC50 value (280nM for H3K36Me2 and > 10µM for H3K36Me1). The kit displayed more than 30 fold selectivity for H3K36Me2 over H3K36Me1

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
H3K36Me2 cellular assay - 10,000 tests62KD2PAD
H3K36Me2 cellular assay - 500 tests62KD2PAE

Companion products

DescriptionCat. noProduct insertMSDS
Epigeneous lysis buffer B - (4X) - 500 tests62EL2FDD
Epigeneous lysis buffer B - (4X) - 10,000 tests62EL2FDH
Epigeneous lysis buffer C - 500 tests62EL3FDD
Epigeneous lysis buffer C - 10,000 tests62EL3FDH
H3K36Me2 cellular control lysate62KD2TDA