The total Histone H3 kit detects the nuclear Histone H3 level and can be used as a normalization assay for the phospho-Histone H3 kit.
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  • Faster and more convenient than ELISA Faster and more convenient than ELISA
The total Histone H3 kit detects the nuclear Histone H3 level and can be used as a normalization assay for the phospho-Histone H3 kit.
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Overview

The Total Histone H3 cellular assay kit is used as a normalization assay with phospho-Histone H3 kits. Histone H3 is a key regulator of gene expression and mitosis, making it a powerful tool for anti-cancer research.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Total-Histone H3 assay principle

The Total-Histone H3 assay quantifies the expression level of Histone H3 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-Histone H3 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of Histone H3 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.
Total Histone H3 cellular assay principle

Total-Histone H3 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Total Histone H3 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total Histone H3 2-plate assay protocol

Total-Histone H3 1-plate assay protocol

Detection of total Histone H3 with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Total Histone H3 1-plate assay protocol

Total-Histone H3 as normalization control for phospho-Histone H3 (Ser10) assay

NIH3T3 cells (12.5KC per well) were seeded in a 96-well plate and treated with Nocodazole, an antimitotic agent that depolymerizes microtubules and blocks the cells in mitosis, to stimulate Histone H3 (Ser10) phosphorylation. Cells were (experiment a) or weren’t (experiment b) co-treated for 16 hours with an inhibitor Danusertib. After cell culture medium removal, 200 µL of supplemented lysis buffer were added for a 30-minute incubation. 10 µL of lysates each were transferred to a 384sv white plate for HTRF analysis in parallel with the total and the phospho-Ser10 assays
Stimulation of Histone H3 phosphorylation by nocodazole
Danusertib inhibitor on Histone H3 phosphorylation after stimulation by nocodazole

Total Histone H3 cell signaling

Histone3 is one of the 5 major canonical Histone proteins to compact DNA into chromatin. The main globular histone domains remain in the long N-terminal Histone tails that are highly post-translationally modified and are responsible for the constitution of either highly condensed heterochromatin or less condensed euchromatin. The different types of coordinated modifications, like acetylation, methylation and phosphorylation, compose the histone code, read by the remodeling machinery. Thus Histone modifications play a major role in the dynamic and long term regulation of genes, making the surrounding DNA more or less accessible to the transcriptional machinery. Phosphorylation of Ser10 or Thr3 in the tails of Histone H3 is involved in gene expression and during mitosis.
total Histone H3 cell signaling pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

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Cisbio lysis buffer compatibility

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Species compatibility

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HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

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Get the brochure about technology comparison. - Brochures

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