AlphaLISA SureFire Ultra Human and Mouse Phospho-AKT (Thr308) Detection Kit, 500 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 enables cell 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 robust AlphaLISA SureFire Ultra quality.

MCF7 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 serum starved for 2 hours and then treated with increasing concentrations of insulin for 10 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AKT1/2/3 Phospho (Thr308) 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, insulin triggered a dose-dependent increase in the levels of AKT1/2/3 Phospho (Thr308) while Total levels remained unchanged.

NIH/3T3 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 serum starved for 2 hours and then treated with increasing concentrations of insulin for 300 minutes.

After treatment, the cells were lysed with 150 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AKT1/2/3 Phospho (Thr308) 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, insulin triggered a dose-dependent increase in the levels of AKT1/2/3 Phospho (Thr308) while Total levels remained unchanged.

Inhibition of AKT1/2/3 Phospho (Thr308) in HEK293 cells

HEK293 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 Luminespib for 24 hours.

After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AKT1/2/3 Phospho (Thr308) 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, Luminespib treatment resulted in a dose-dependent decrease in the levels of AKT1/2/3 Phospho (Thr308) and a modest decrease in Total signal.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from HEK293 cells cultured to confluence in T175 flasks and treated with 20% FBS for 15 minutes. Cells were lysed with 4 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysates were serially diluted in Lysis Buffer and AKT1/2/3 Phospho (Thr308) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. 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/datapoint is indicated on the graph. The dotted line represents assay background. This assay can detect AKT1/2/3 Phospho (Thr308) in less than 500 cells/datapoint.