AlphaLISA SureFire Ultra Human Total AKT1/2/3 Detection Kit, 500 Assay Points

Validation of AKT1/2/3 in insulin treated cells

HeLa 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 serum starved for 3 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). AKT1/2/3 Phospho (Ser473) 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 an increase in the levels of Phospho (Ser473) AKT1/2/3 while Total levels remained unchanged.

Validation of AKT1/2/3 in Razuprotafib treated cells

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 serum 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). AKT1/2/3 Phospho (Ser473) 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 an increase in level of Phospho (Ser473) AKT1/2/3 while Total levels remained unchanged.

Validation of AKT1/2/3 in Wortmannin 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 treated 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). AKT1/2/3 Phospho (Ser473) 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, Wortmannin triggered a decrease in the levels of Phospho (Ser473) AKT1/2/3 while Total levels remained unchanged.

Validation of AKT1/2/3 phospho-(Ser473)/AKT1/2/3 total in Torin1 treated cells

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 treated with increasing concentrations of Torin1 for 18 hours.

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 (Ser473) 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, Torin1 triggered a decrease in level of Phospho (Ser473) AKT1/2/3 while Total AKT1/2/3 levels remained unchanged.

Degradation of AKT1/2/3 total in PROTAC treated cells

PC3 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 AKT PROTAC, MS170 for 24 hours. After treatment, the cells were washed with HBSS and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AKT1/2/3 Total and ERK1/2 Total levels were evaluated using respective ALphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 6,000 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 AKT PROTAC MS170 triggered a dose-dependent decrease in the levels of Total AKT1/2/3 while Total ERK1/2 levels remain unchanged. 

AKT1/2/3 Total assay specificity

Specificity of the AKT1/2/3 Total assay was assessed by using AKT1, 2 and 3 proteins.

Dilutions of recombinant AKT1 (Abcam, ab62279), AKT 2 (Abcam, ab268317) and AKT3 (Abcam, ab60324) were prepared in Lysis Buffer and evaluated using the AlphaLISA SureFire Ultra assay.

For the detection step, 10 µL of diluted protein 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.

The AKT1/2/3 Total assay shows reactivity to all three AKT isoforms.