Phospho-Histone H3 (Thr3) cellular kit HTRF®

The phospho-Histone H3 (Thr3) assay enables the cell-based detection of Thr3 phosphorylation of Histone H3, a key regulator of gene expression and mitosis.
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The phospho-Histone H3 (Thr3) assay enables the cell-based detection of Thr3 phosphorylation of Histone H3, a key regulator of gene expression and mitosis.


Phospho-Histone H3 (Thr3) assay enables the detection of Thr3 phosphorylation in the tails of Histone H3, a key regulator of gene expression and mitosis. Phosphorylation at Threonine 3, catalyzed by the kinase haspin and highly conserved among many species, leads to recruitment of Aurora kinase B (AURKB) and the chromosomal passenger complex (CPC) to kinetochores, to regulate mitosis.



Phospho-Histone H3 (Thr3) assay principle

The phospho-Histone H3 (Thr3) assay measures Histone H3 when phosphorylated at Thr3. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The phospho-Histone H3 (Thr3) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.

Phospho-Histone H3 (Thr3) assay principle

Phospho-Histone H3 (Thr3) 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 phospho-Histone H3 (Thr3) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-Histone H3 (Thr3) 2-plate assay protocol

Phospho-Histone H3 (Thr3) 1-plate assay protocol

Detection of Phosphorylated Histone H3 (Thr3) 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.
Phospho-Histone H3 (Thr3) 1-plate protocol

HTRF phospho H3 (Thr3) assay vs Western Blot

Human cervical cancer HeLa cells were cultured until reaching 80% confluency. After treatment with Calyculin A, cells were lysed and soluble fractions collected after a 10 min centrifugation. Serial dilutions of the cell lysate were performed, dispensed and analyzed side-by-side by Western Blot and by HTRF. The HTRF phospho H3 (Thr3) assay is at least 16-fold more sensitive than Western Blot.
HTRF phospho H3 (Thr3) assay vs Western Blot

HTRF phospho H3 (Thr3) validation assay on mouse and human cells

50,000 human cervical cancer HeLa cells or mouse fibroblast NIH-3T3 cells were plated in 96-well plates and incubated for 24h at 37 °C - 5% CO2. Cells were stimulated at 37°C for 30 min with increasing concentration of Calyculin-A. Medium was then removed and cells were lysed with 50µl of lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate and 4 µL of the HTRF phospho-Histone H3 (Thr3) detection reagents were added. The HTRF signal was recorded after a 2h incubation time at room temperature.
HTRF phospho H3 (Thr3) validation assay on mouse NIH3T3 cells
HTRF phospho H3 (Thr3) validation assay on human HeLa cells

Phospho-Histone H3-Thr3 in cell signaling

There are four characterized residues within the H3 N-terminal tail that are phosphorylated during mitosis: Thr3, Ser10, Thr11, and Ser28. There is a precise spatio-temporal correlation between Thr3 phosphorylation and stages of mitosis: the phosphorylation starts at early prophase in the vicinity of the nuclear envelope, spreads to pericentromeric chromatin during prometaphase and is fully reversed by late anaphase. Phosphorylation at Thr3 is highly conserved among many species and is catalyzed by the kinase Haspin. Thr3 phosphorylation then provides a chromatin binding site for the recruitment of the chromosomal passenger complex (CPC) which consists of four proteins: Survivin which binds to H3, Aurora-B, INCEMP and Borealin. The Aurora kinase B then phosphorylates Haspin, which creates a signal amplification loop and contributes to CPC accumulation at centromeres to regulate mitosis and chromosome segregation.The dephosphorylation is catalyzed by the complex PP1-qamma/Repo-Ma.

Pathway of Phospho-Histone H3-Thr3 in cell signaling

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

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

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

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

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