Revvity, 64H2AXTPEG, HTRF Human Total H2AX Detection Kit, 500 Assay Points

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.
How it works
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.

 

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. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 3 Lysis Buffer 4
Molecular Modification-Total
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Unit Size-500 assay points