AlphaLISA SureFire Ultra Human Phospho-PI3K p85 (Tyr467) Detection Kit, 10,000 Assay Points

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.

Activation of Phospho PI3K (Tyr467) levels in Insulin treated cells

HEK293 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were starved for 2 hours and then treated with increasing concentrations of Insulin for 5 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). PI3K Phospho (Tyr467) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 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, Insulin triggered a dose-dependent increase in the levels of Phospho PI3K (Tyr467) while Total PI3K levels remained unchanged.

Decrease of Phospho PI3K (Tyr467) levels in Luminespib treated cells

HEK293 cells were seeded in a 96-well plate (60,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of Luminespib for 24 hours.

After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). PI3K Phospho (Tyr467) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 12,000 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.

Luminespib treatment in HEK293 cells triggered a significant dose-dependent decrease in the levels of Phospho PI3K (Tyr467) while PI3K Total levels remain unchanged.

Decrease of Phospho PI3K (Tyr467) levels in Dasatinib treated cells

HEK293 cells were seeded in a 96-well plate (60,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of Dasatinib for 24 hours.

After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). PI3K Phospho (Tyr467) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 12,000 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.

Dasatinib treatment in HEK293 cells triggered a dose-dependent decrease in the levels of Phospho PI3K (Tyr467) while PI3K Total levels remain unchanged.