-->

Phospho-Histone H3 (Ser10) cellular kit HTRF®

The phospho-Histone H3 (Ser10) assay enables the cell-based detection of Ser10 phosphorylation of Histone H3, a key regulator of gene expression and mitosis.
See more
  • Ready-to-use Ready-to-use
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
The phospho-Histone H3 (Ser10) assay enables the cell-based detection of Ser10 phosphorylation of Histone H3, a key regulator of gene expression and mitosis.
-

Overview

The Phospho-Histone H3 (Ser10) assay enables the detection of Ser10 phosphorylation in the tails of Histone H3, a key regulator of gene expression and mitosis. Phosphorylation at Ser10 is tightly correlated with chromosome condensation during both mitosis and meiosis.

Benefits

  • SPECIFICITY
  • PRECISION

Phospho-Histone H3 (Ser10) assay principle

The phospho-Histone H3 (Ser10) assay measures Histone H3 when phosphorylated at Ser10. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The phospho-Histone H3 (Ser10) 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 (Ser10) assay principle

Phospho-Histone H3 (Ser10) 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 (Ser10) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-Histone H3 (Ser10) 2-plate assay protocol

Phospho-Histone H3 (Ser10) 1-plate assay protocol

Detection of Phosphorylated Histone H3 (Ser10) 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 (Ser10) 1-plate protocol

HTRF phospho-Histone H3 (Ser10) assay compared to Western Blot

Exponentially growing Hela cells were cultured to 80% confluency and treated with Nocodazole for 16 hours. After medium removal and lysis, soluble supernatants were collected via centrifugation. Equal amounts of lysates were used for a side by side comparison of WB and HTRF. The HTRF assay is 25-fold more sensitive than the Western Blot. Using HTRF phospho-Histone H3 (Ser10), only 256 cells are sufficient for minimal signal detection, while 6,400 cells are needed for a Western Blot signal. The HTRF assay is 25-fold more sensitive than the Western Blot.
HTRF phospho-Ser10 assay compared to Western Blot

Phospho-Histone H3 (Ser10) as a mitotic biomarker for cell proliferation

Hela and A431 cells were seeded in a 96-well plate and treated for 16 hours with Nocodazole, an antimitotic agent that depolymerizes microtubules and blocks the cells in mitosis, when Histone H3 Ser10 phosphorylation is maximal. After cell culture media removal, 50 µl, 100 µl or 200 µl of lysis buffer were added for a 30-minute incubation. 16 µl of each lysate were transferred to a 384sv white plate for HTRF analysis. Increasing the lysis volume up to 200µl improved the linearity of the HTRF detection signal.
Histone H3 (Ser10) phosphorylation stimulation by nocodazole in HeLa cells
Histone H3 (Ser10) phosphorylation inhibition by danusetib in nocodazole-stimulated A431 cells

Phospho-Histone H3 (Ser10) as read-out for Aurora Kinase B inhibition

Histone H3 (Ser10) phosphorylation inhibition by danusertib in HeLa cells
Histone H3 (S10) phosphorylation inhibition by danusertib in Jurkat cells
Histone H3 (S10) phosphorylation inhibition alisertib in NIH3T3 cells

Phospho-Histone H3-Ser10 in the cell cycle

Overall histone phosphorylation fluctuates very markedly during the cell cycle and is characterized by peaks in the phosphorylation of Histone H3 on Serine10 in mitosis. Aurora kinase B is directly responsible for the mitotic Histone H3 Ser10 phosphorylation. Aurora kinase A, in complex with BORA, is responsible for the initial activation of PLK1 in G2 phase, leading to the activation of cyclin-dependent kinase 1 mitotic entry at the G2/M transition checkpoint. Aurora kinase A is frequently amplified in human cancers, and Aurora kinase A overexpression in human tumours correlates with a poor prognosis and genomic instability. Moreover, high expression levels of Aurora kinase B are associated with a poor prognosis in glioblastoma, ovarian carcinoma and hepatocellular carcinoma.
Phospho-Histone H3-Ser10 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.

Let's find your reader