Description
H2A histone family member X (H2AX) is a type of histone protein encoded by the H2AFX gene. H2AX phosphorylation on serine 139 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. Analyzing H2AX allows for a better understanding of genome stability, cell cycle, DNA repair, and more broadly, H2AX is a biomarker for cancer and for aging process investigations.
The AlphaLISA SureFire Ultra Multiplex Human Phospho-H2A.X (Ser139)/Total H2A.X Detection Kit is used to measure phosphorylated H2A.X (Ser139) and total levels of H2A.X in cellular lysates in a multiplexing Alpha no-wash assay. No specially engineered cell lines are required. The 615 nm (Eu) signal corresponds to the phosphorylated H2A.X, and the 545 nm (Tb) signal corresponds to the total H2A.X analysis. This kit has been formulated to provide superior signal:background assay windows and to perform without interference in the presence of extraneous antibodies, making it amenable to the study of therapeutic and blocking antibodies.
No-wash steps, no separation steps
Multiplexing assay
Sensitive detection
No interference from exogenous antibodies
Results in less than 3 hours
In this assay, the Alpha 615 Acceptor bead is coated with the CaptSure™ antibody, which binds the CaptSure-tagged anti-phospho target antibody. The Alpha 545 Acceptor bead is coated with the CaptSure2™ agent, which binds the CaptSure2 tagged anti-total target protein antibody. The Alpha Donor bead binds the biotinylated anti-total target protein antibody.
Multiplex SureFire Ultra assay principle
The Multiplex SureFire Ultra assay simultaneously measures phospho and total protein at a specific level in a biological sample (e.g. cell lysate). It utilizes three antibodies that recognize the phospho epitope and two distal epitopes on the target protein. The assay requires three bead types: two distinct Acceptor Beads and one Donor Bead.
The Alpha 615 Acceptor Bead is coated with the CaptSure™ antibody, which binds the CaptSure-tagged anti-phospho target antibody.
The Alpha 545 Acceptor Bead is coated with the CaptSure2 antibody, which binds the CaptSure2-tagged anti-total target protein antibody.
The Alpha Donor Bead binds the biotinylated anti-total target protein antibody.
When the phosphorylated protein is present, the three antibodies bring the Donor and Acceptor Beads close together, allowing singlet oxygen to transfer energy and excite both Acceptor Beads. This generates a luminescent Alpha signal at 615nm and 545nm. The 615nm (Eu) signal is directly proportional to the amount of phosphorylated protein, while the 545nm (Tb) signal corresponds to the total protein levels.
Multiplex SureFire Ultra two-plate assay protocol
The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Multiplex SureFire Ultra detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure different targets.
Multiplex SureFire Ultra one-plate assay protocol
Detection of Phosphorylated/Total target proteins with Multiplex SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire Ultra quality.
