AlphaLISA SureFire Ultra Human Total BAX Detection Kit, 10,000 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

U2OS cells were seeded in a 96-well plate (10,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of etoposide for 24 hours in serum-free media.

After treatment, cells washed with HBSS and lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Total Bax and Cofilin levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 1,000 cells or 200 cells for Cofilin) 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 significant increase in the levels of Total Bax whilst Total Cofilin remained unchanged.

Knockout validation of Bax Total assay

Bax Total levels were assessed in HeLa Wild Type (WT) and Bax knockout (KO) cells (Abcam ab255363). Cells were seeded at various densities in a 96 well plate in complete medium and incubated overnight at 37°C, 5% CO₂.

The cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Bax levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate 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 Total signal was only detected in the WT cells confirming the specificity of the assay.

Expression of Bax in various human cell lines

Adherent 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 200 µL of Lysis Buffer.

Suspension cells were harvested, washed with HBSS and lysed at various cell densities. Cell lysates were diluted to 1 x 10⁶ cells/mL with Lysis Buffer.

Bax Total levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 4,000 adherent cells or 10,000 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 a wide range of human adherent and suspension cell lines.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from A549 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 50 cells/datapoint.