AlphaLISA SureFire Ultra Mouse Total BAX Detection Kit, 500 Assay Points

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 permits the cells 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 AlphaLISA SureFire Ultra quality.

Induction of Bax in etoposide treated cells

EL-4 cells were seeded in a 96-well plate (100,000 cells/well) and treated with increasing concentrations of etoposide in serum free-media for 24 hours at 37°C, 5% CO₂.

After treatment, the cells were washed and resuspended in 200 µL HBSS. Cells were lysed with 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Bax and Cofilin Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) 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, etoposide triggered a dose-dependent increase in the levels of Total Bax while Cofilin Total remained unchanged.

Assay versatility - cell panel

Cells were seeded at 40,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were lysed with 100 µL of Lysis Buffer.

Suspension cells were seeded at 400,000 cells/well in a 96-well culture plate in HBSS + 0.1% BSA, cells were spun down and lysed with 100 µL of Lysis Buffer. Cell lysates were further diluted in Lysis Buffer. Bax Total levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 1,000 adherent cells or 5,000 suspension cells) 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.

Bax expression was detected in various mouse adherent and suspension cell lines.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from C2C12 cells cultured to confluency in a T175 flask and lysed in 16 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and Total Bax levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. 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.

Approximate number of cells per datapoint is indicated. The dotted line represents assay background. The assay can detect Total Bax down to 100 cells/datapoint.