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.

Pharmacological validation with Neocarzinostatin compound

HEK293 cells were cultured in a 96-well plate (12,500 cells/well) for 24 hours at 37°C, 5% CO2. After cell culture medium removal, they were treated with increasing concentrations of Neocazinostatin (DNA damaging agent). After a 30 min incubation, the cell culture medium was removed and 50 µl of supplemented Lysis Buffer#4 (1X) were dispensed into each well. After 45 min of cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total H2AX or Phospho-H2AX (Ser139) detection antibodies were added. The HTRF signal was recorded after a 2h incubation.

The Neocazinostatin triggered a dose-dependent increase in Phosphorylated H2AX (Ser139) protein, while the expression level of H2AX remained stable.

Specificity of HTRF Total H2AX assay using knockout H2AX cell lines

The Human H2AX expression level was assessed with the HTRF Total H2AX kit in HAP1 cells (WT) and HAP1 knocked-out (KO) for H2AX. The cell density was optimized beforehand to ensure HTRF detection within the dynamic range of the kit (data not shown).

The cell lines were cultured in a 96-well plate (50,000 cells/well) for 24 hours at 37°C, 5% CO2. After culture medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #4 (1X) for 45 min, then 16 µL of cell lysate were transferred into a low volume white microplate followed by 4 µL of premixed detection reagents. The HTRF signal was recorded after a 2h incubation at RT.

In HAP1 KO H2AX cells, the HTRF signal was equivalent to the non-specific signal (dotted line), indicating a complete H2AX gene silencing and demonstrating the specificity of the HTRF Total H2AX kit.

HAP1 Ko cell line from Horizon Discovery # HZGHC005630c003

HAP1 Wt from Horizon Discovery # C631