Total Histone H3 cellular kit HTRF®

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.


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.



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

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

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

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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