H2AX is a histone variant that comprises approximately 10% of total H2A in mammalian cells and serves as a critical sensor and mediator of DNA double-strand break responses. Upon DNA damage, H2AX is rapidly phosphorylated at Ser139 by ATM, ATR, and DNA-PK kinases, forming γH2AX foci that spread megabases from the break site. γH2AX serves as a platform for recruiting DNA repair factors including MDC1, 53BP1, and BRCA1, facilitating homologous recombination and non-homologous end joining. The formation of γH2AX foci is widely used as a biomarker for DNA damage and genotoxic stress in research and clinical settings. Loss of H2AX function leads to genomic instability, radiation sensitivity, and increased cancer susceptibility, while persistent γH2AX accumulation indicates defective DNA repair or replicative stress in cancer cells.
The AlphaLISA SureFire Ultra Mouse Total H2AX Detection Kit is a sandwich immunoassay for the quantitative detection of total H2AX in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
AlphaLISA SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Total-AlphaLISA SureFire Ultra assay principle
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of target protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.
Total-AlphaLISA 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 Total-AlphaLISA 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 multiple targets.
Total-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of Total target protein with AlphaLISA 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.
Validation of Histone H2A.X Total in cells treated with bleomycin
NIH/3T3 and RAW 264.7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. Cells were treated with increasing concentrations of bleomycin for 2 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Histone H2A.X Phospho (Ser139) and Total levels were evaluated using AlphaLISA SureFire assays. For the detection step, 10 µL of cell lysate (approximately 1,000 cells for Phospho, 4,000 cells for Total) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, bleomycin triggered a dose-dependent increase in the levels of Histone H2A.X (Ser139) phosphorylation, while a modest decrease was observed in Histone H2A.X Total levels.
EL4 cells were seeded in a 96-well plate (50,000 cells/well) in complete medium and treated with increasing concentrations of bleomycin for 2 hours.
After treatment, the cells were washed with HBSS and lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Histone H2A.X Phospho (Ser139) and Total levels were evaluated using AlphaLISA SureFire assays. For the detection step, 10 µL of cell lysate (approximately 1,250 cells for Phospho, 2,500 cells for Total) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, bleomycin triggered a dose-dependent increase in the levels of Histone H2A.X (Ser139) phosphorylation, while Total levels remained unchanged.
Histone H2A.X expression in various cell lines
Adherent cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. Cells were lysed with Lysis Buffer at a density of 40K cells/mL. Suspension cells were harvested, seeded in a 96-well plate (50,000 cells/well), washed in HBSS and lysed with Lysis Buffer at a density of 250K cells/mL.
Histone H2A.X Total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 4,000 adherent cells and 2,500 suspension cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
Total Histone H2A.X expression was detected in a wide range of mouse cell lines.
