Revvity, 64H2XPEH, HTRF Human Phospho-H2AX (Ser139) Detection Kit, 10,000 Assay Points

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.
How it works
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 proteins phosphorylation state under a no-wash assay format.

 

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) 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. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 4 Lysis Buffer 5
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Therapeutic Area-Oncology & Inflammation
Unit Size-10,000 assay points