EPIgeneous™ Total H3 Cellular Assay Kit

Total H3 Cellular assay for studying methyltransferases and demethylases

This cellular assay is for the direct detection of endogenous levels of Total H3 (histone 3) in cells. It has been developed with optimized reagents and protocols. HTRF® is a flexible assay format enabling you to use an assay for a variety of applications, without having to compromise on sensitivity.  The Total H3 assay can be used for adherent cells or suspension cells, primary or secondary screening, inhibitor studies and normalization studies of both methyltransferases and demethylases. The total H3 kit has been validated with Cisbio’s EPIgeneous H3K27ME3 and H3K36ME2 kits for normalization studies.

Assay principle

This assay is designed for the simple, rapid and direct detection of endogenous levels of H3 Total in cells. 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). The specific signal modulates positively in proportion to H3 Total presence.

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 Total H3 by HTRF reagents. This protocol allows the cells' viability and confluence to be monitored. Moreover, the Total H3 kit has been designed to be run in parallel with our EPIgeneous specific methyl mark cellular assay. With this, a high lysis volume makes it possible to assay both methyl mark and H3 total from the same 96w lysate.

One-plate assay protocol

Detection of Total H3 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 Total Histone H3

HTRF Total Histone H3 detection in various cell lines from different species (human, hamster and mouse) and formats (suspension or adherent cells). The numbers of cells indicated on the graph represent the final number of cells per well in the 384w plate. Cells were lysed using Lysis Buffer A.

Normalization

HTRF detection of H3K27Me3 and total Histone H3 in SU-DHL6 and OCI LY 19 cell lines. Cells were seeded at 20 000cells/w in 96w plate and treated for 72h with selective EZH2 inhibitor GSK126 (from 10µM to 13.7nM diluted in medium). Using the two plate protocol, each cell lysate from each condition could be assayed on both methyl mark (here H3K27Me3) and total Histone H3, in order to measure the specificity of the acting compound.

The compound tested here showed no effect on Total Histone H3 below 3.33µM, whereas it fully inhibited the signal on H3K27Me3. 10µM of GSK126 clearly creates an inhibition on Total H3 detection.

DMSO Sensitivity

HTRF Total Histone H3 detection in HeLa cells. Cells were seeded at various densities in a 96w plate and incubated for 72h before lysis with lysis Buffer B (lysis step without discarding medium). Different DMSO concentrations (0%, 0.5%, 1%) were added to a 96w plate containing  HeLa cells. the assay fully demonstrates tolerance to DMSO.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Total H3 cellular assay 10,000 tests62NH3PAD
Total H3 cellular assay - 500 tests62NH3PAE

Companion products

DescriptionCat. noProduct insertMSDS
Total H3 cellular control lysate62NH3TDA
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