AlphaLISA SureFire Ultra Human and Mouse Phospho-PRAS40 (Thr246) Detection Kit, 100 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 PRAS40 (Thr246) in Insulin treated cells

HeLa cells were seeded in a 96-well plate (20,000 cells/well) in complete medium, and incubated over 48 hours at 37°C, 5% CO2. The cells were starved for 3 hours 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). PRAS40 Phospho (Thr246) 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 PRAS40 (Thr246) while Total PRAS40 levels remained unchanged.

Decrease of Phospho PRAS40 (Thr246) in UCN-01 treated cells

A549 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of UCN-01 for 1 hour.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). PRAS40 Phospho (Thr246) and 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, UCN-01 triggered a dose-dependent decrease in the levels of Phospho PRAS40 (Thr246) while Total PRAS40 levels remained unchanged.

Decrease of Phospho PRAS40 (Thr246) in Staurosporine treated cells

A549 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of Staurosporine for 1 hour.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). PRAS40 Phospho (Thr246) and 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, Staurosporine triggered a dose-dependent decrease in the levels of Phospho (Thr246) PRAS40 while Total PRAS40 levels remained unchanged.

Decrease of Phospho PRAS40 (Thr246) in Wortmannin treated cells

A549 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of Wortmannin for 1 hour.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). PRAS40 Phospho (Thr246) and 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, Wortmannin triggered a dose-dependent decrease in the levels of Phospho PRAS40 (Thr246) while Total PRAS40 levels remained unchanged.