AlphaLISA SureFire Ultra Human and Mouse Phospho-AKT3 (Ser472) 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.

AKT3 phosphorylation induced by TIE2 pathway activation

HUVEC 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 Razuprotafib for 15 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AKT3 Phospho (Ser472) 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, Razuprotafib triggered a dose-dependent increase in the levels of AKT3 Phospho (Ser472) while total levels remained unchanged.

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 for 24 hours 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). AKT3 Phospho (Ser472) 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 triggered a dose-dependent decrease in the levels of AKT3 Phospho (Ser472) while total levels remained unchanged.

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 for 2 hours with increasing concentrations of Wortmannin for 2 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AKT3 Phospho (Ser472) 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 AKT3 Phospho (Ser472) while total levels did not change significantly.

Specificity of AKT3 Phospho (Ser472) assay

Specificity of the AKT3 Phospho (Ser472) assay for AKT3 protein was assessed by assaying active recombinant AKT1, AKT2 and AKT3 proteins.

Dilutions of AKT1 (Abcam, ab62279), AKT2 (Abcam, ab60324) and AKT3 (Abcam, ab60324) proteins were prepared in Lysis Buffer at the indicated concentrations. AKT3 Phospho (Ser472) signal was evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of protein 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.

Alpha signal was only detected with AKT3 recombinant protein, while no cross-reactivity was observed with AKT1 and AKT2 proteins. These results demonstrate the specificity of the AKT3 Phospho assay as these three proteins share approximately 80% sequence identity.