Phospho-H2AX (Ser139) Cellular Assay Kit

Sensitive phospho-H2AX detection, as a biomarker for DNA repair of double-strand breaks

Cisbio's cell-based homogeneous HTRF® phospho-H2AX Ser139 assay enables the quantitative detection of the histone variant H2AX, phosphorylated on serine 139. When double-stranded DNA breaks, either by ionizing radiation or endogenous physiological processes, H2AX becomes phosphorylated at Ser139. This modification, called gamma-H2AX (phospho-Ser139), serves as a sensitive biomarker for the detection of such breaks, localizing the site of DNA repair. Gamma-H2AX triggers cell cycle arrest and DNA damage response repair, which makes HTRF® phospho-H2AX Ser139 a pertinent assay to study genome stability, the cell cycle, DNA repair, and it serves as a biomarker for cancer, and to understand aging. The simple add-and-read protocol eliminates all wash steps for a faster analysis and permits easy miniaturization. The assay is amenable to low-volume formats for all throughputs and offers enhanced convenience over other immunoassay technologies.

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Assay Principle

HTRF® - the homogeneous cell-based sandwich immunoassay

The phospho H2AX assay is based on a TR-FRET sandwich immunoassay format comprising two specific, monoclonal anti-H2AX antibodies, one labeled with Europium-cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind H2AX, and the proximity of donor and acceptor will then lead to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho-H2AX can be quantitatively detected using the HTRF phospho-H2AX (Ser139) cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384sv assay plate where the HTRF® reagents are added. This also enables the cells' viability and confluence to be monitored in an appropriate cell culture plate.

One-plate assay protocol

This protocol can be further streamlined to a one-plate assay in which plating, treating and detecting are all performed in a single plate. No wash steps are required. Ideal for HTS - this protocol provides enhanced speed and simplicity, enabling all throughputs and fast results while maintaining a high-quality sensitive output.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Simplified pathway

Gamma-H2AX signaling in DNA double strand break induced DNA damage response

H2AX is a variant of the histone H2A family, which is a component of the histone octamer in nucleosomes. DNA Double-Strand Breaks (DSB) are the most deleterious type of DNA damage, occurring due to endogenous processes or exogenous factors (e.g. ionizing & UV radiations, specific anticancer drugs). When a DSB takes place, the tri-protein MRN complex (MRE11-RAD50-NBS1) recognizes the DNA damage and recruits and activates the PI3-kinase related kinase ATM (Ataxia Telangiectasia Mutated). ATM phosphorylates H2AX on its c-terminal Ser139 residue. This modification is called γ-H2AX and is spread within minutes to thousands of H2AX proteins that are in proximity to the damage site. This phosphorylation of H2AX on Ser139 is crucial to activating the DNA damage response pathway, a complex molecular mechanism to detect and repair DNA damage. γ-H2AX then attracts the Mediator of Damage Checkpoint protein 1 (MDC1), which is also phosphorylated by ATM. MDC1 in turn serves as a scaffold for the recruitment of other proteins required for the activation of BRCA1 by ATM, promoting cell cycle arrest and DNA repair. ATM phosphorylates other target substrates like the checkpoint protein Chk2 and p53, which are also responsible for cell cycle arrest or apoptosis if the damage cannot be repaired. After the DNA has been repaired, γ-H2AX is dephosphorylated by the phosphatase PP2A in human cells. The efficient repair of DSBs is essential to maintain genome stability and cell viability. Unrepaired or misrepaired DSBs can initiate processes leading to mutagenesis, tumorigenesis and apoptosis.

Product Performance

1. HTRF assay compared to Western Blot using phospho-H2AX cellular assays

HEK-293 cells were grown in a T175 flask at 37 °C, 5% CO2 until 80% confluency. Cell culture medium was then discarded and cells were lysed with 3 mL of supplemented lysis buffer for 30 min at room temperature. Soluble fractions were then collected after a 10 min 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.

Using HTRF phospho-H2AX (Ser139) cellular assay, only 2,500 cells are sufficient for minimal signal detection while 40,000 cells are needed for a Western Blot signal. The HTRF cellular assays are at least 16-fold more sensitive than the Western Blot.

2. Neocarzinostatin dose-response on Jurkat cells using phospho-H2AX(Ser139) cellular assay

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.

3. Phospho-H2AX(Ser139) activation on Jurkat cells: kinetic of detection

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.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
H2AX phospho-S139 kit - 500 tests64H2XPEG
H2AX phospho-S139 kit - 10,000 tests64H2XPEH
H2AX phospho-S139 kit - 50,000 tests64H2XPEY
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Companion products

DescriptionCat. noProduct insertMSDS
H2AX phospho-S139 kit control lysate64H2XTDA
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Phospho-total protein blocking reagent - 2 ml64KB1AAC
Phospho-total protein blocking reagent - 6 ml64KB1AAD
Phospho-total protein lysis buffer #4 - 130 mL64KL4FDF

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