The phospho-H2AX (Ser139) kit enables the quantitative cell-based detection of the histone variant H2AX, phosphorylated on serine 139.
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  • Ease-of-use Ease-of-use
  • Highly specific Highly specific
The phospho-H2AX (Ser139) kit enables the quantitative cell-based detection of the histone variant H2AX, phosphorylated on serine 139.
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Overview

The phospho-H2AX (Ser139) kit offers quantitative detection of the histone variant H2AX, phosphorylated on serine 139. This specific phosphorylation may occur when double-stranded DNA breaks, either due to ionizing radiation or to endogenous physiological processes. This modification, called gamma-H2AX (phospho-Ser139), triggers cell cycle arrest and DNA damage response repair, which makes this assay particularly appropriate for studying genome stability, cell cycle, DNA repair, and more broadly as a biomarker for cancer and for aging process investigations.

Benefits

  • SPECIFICITY
  • PRECISION

Phospho-H2AX (Ser139) assay principle

The Phospho-H2AX (Ser139) assay measures H2AX when phosphorylated at Ser139. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-H2AX (Ser139) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is specific for the phosphorylated motif on the protein, the second one recognizes 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-H2AX (Ser139) assay principle

Phospho-H2AX (Ser139) 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-H2AX (Ser139) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-H2AX (Ser139) 2-plate assay protocol

Phospho-H2AX (Ser139) 1-plate assay protocol

Detection of Phosphorylated H2AX (Ser139) 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-H2AX (Ser139) 1-plate assay protocol

HTRF phospho-H2AX cellular assay compared to Western Blot

HEK-293 cells were grown in a T175 flask to 80% confluency. Cell culture were then lysed and pelleted by centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were dispensed and analyzed side-by-side by Western Blot and by HTRF. The HTRF cellular assays were found to be at least 16-fold more sensitive than the Western Blot.
HTRF phospho-H2AX cellular assay compared to Western Blot

Neocarzinostatin dose-response on Jurkat cells

25,000 human Jurkat cells were plated in 96 well plates. After incubation with increasing concentrations of neocarzinostatin (30min), cells were lysed with 10µl of lysis buffer 4X 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-H2AX (Ser139) detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Neocarzinostatin dose-response on Jurkat cells

Phospho-H2AX(Ser139) activation on Jurkat cells: detection kinetics

25,000 human Jurkat cells were plated in 96 well plates. After incubation with increasing concentrations of neocarzinostatin (30min), cells were lysed with 10µl of lysis buffer 4X for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well small volume white microplate and 4 µL of the HTRF phospho-H2AX (Ser139) detection reagents were added. The HTRF signal was recorded after different incubation times.
Phospho-H2AX(Ser139) activation on Jurkat cells: detection kinetics

Phospho-H2AX (Ser139) cellular assay kit simplified pathway

DNA Double-Strand Breaks (DSB) are the most deleterious type of DNA damage, consecutive to endogenous processes or exogenous factors (e.g. ionizing & UV radiations, specific anticancer drugs). H2AX is a variant of the histone H2A family, which gets phosphorylated at Ser139 upon the event of a DSB. This modification is called ?-H2AX and is spread within minutes to thousands of H2AX proteins in proximity to the damage site. It is crucial is crucial to activating the DNA damage response pathway, a complex molecular mechanism to detect and repair DNA damage.
Phospho-H2AX signaling pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

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

Cisbio lysis buffer compatibility

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

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

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

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

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

Product Insert H2AX P-S139 Kit / 64H2XPEG-64H2XPEH

64H2XPEG-64H2XPEH - Product Insert

Cisbio Product Catalog 2019

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

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