AlphaLISA SureFire Ultra Mouse Phospho-H2AX Ser139 Detection Kit, 100 Assay Points

Phospho-AlphaLISA SureFire Ultra assay principle

The Phospho-AlphaLISA SureFire Ultra assay measures a protein target when phosphorylated at a specific residue.

The assay uses two antibodies which recognize the phospho epitope and a distal epitope on the targeted 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 phosphorylated 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 the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of phosphoprotein present in the sample.

Phospho-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 Phospho-AlphaLISA SureFire Ultra detection reagents. This protocol permits the cells viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

Phospho-AlphaLISA SureFire Ultra one-plate assay protocol

Detection of Phosphorylated 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 AlphaLISA SureFire Ultra quality.

Induction of Histone H2A.X phosphorylation in various cell models

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.