HTRF Human Total H2AX Detection Kit HTRF®

Associated products

The Total H2AX kit is designed to monitor the expression level of cellular human H2AX, and can be used as a normalization assay for the Phospho-H2AX (Ser139) Detection kit.

All inclusive kit
Low sample consumption
No-wash
High sensitivity

Download the protocols

The Total H2AX kit is designed to monitor the expression level of cellular human H2AX, and can be used as a normalization assay for the Phospho-H2AX (Ser139) Detection kit.

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Overview

The Total-H2AX kit offers quantitative detection of human histone variant H2AX. The specific phosphorylation of H2AX at Ser139 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.

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Total H2AX assay principle

The Total-H2AX assay quantifies the expression level of H2AX in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-H2AX 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 H2AX 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-H2AX 2-plate assay protocol

The 2-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of the Total H2AX HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF total H2AX assay

Total-H2AX 1-plate assay protocol

Detection of Total H2AX 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.

One-plate protocol of the HTRF total H2AX assay

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Product Insert H2AX total Kit / 64H2AXTPEG-64H2AXTPEH

64H2AXTPEG-64H2AXTPEH - Product Insert

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