The Total H3 cellular assay is optimized for the detection of endogeneous levels of Histone H3 in cells.
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  • Cell-based
  • High sensitivity
The Total H3 cellular assay is optimized for the detection of endogeneous levels of Histone H3 in cells.
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Overview

This cellular assay is for the direct detection of endogenous levels of Total H3 (histone 3) in cells. The 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 and be applied to the same assay lysates.

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 Eu-cryptate (donor) and the second with d2 (acceptor). The HTRF signal is proportional to the concentration of histone H3.

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 assess both methylation marks and total H3 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.

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 with Total H3 assay

HTRF detection of H3K27Me3 and total Histone H3 in SU-DHL6 and OCI LY 19 cell lines using a two plate protocol. The test was run to assess the specificity of the acting compound.

Cells were seeded at 20.000cells per well in 96 well plate and treated for 72h with selective EZH2 inhibitor GSK126 (10µM down to 13.7nM diluted in medium).

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.
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