Proline-rich AKT substrate 40 kDa (PRAS40) is a substrate of AKT kinase and a component of the mechanistic target of rapamycin complex 1 (mTORC1). Phosphorylation of PRAS40 by AKT relieves its inhibitory effect on mTORC1, thereby promoting cell growth and proliferation. Dysregulation of PRAS40 phosphorylation is implicated in various cancers and metabolic disorders, highlighting its role in cell survival and metabolism.
The AlphaLISA SureFire Ultra Human and Mouse Total PRAS40 Detection Kit is a sandwich immunoassay for the quantitative detection of total PRAS40 in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Alpha SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
Screening
Total-AlphaLISA SureFire Ultra assay principle
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of target protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.
Total-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 Total-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.
Total-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of Total 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.
Validation of Phospho (Thr246)/total PRAS40 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.
Validation of Phospho (Thr246)/total PRAS40 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 for 1 hour with increasing concentrations of UCN-01.
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.
Validation of Phospho (Thr246)/total PRAS40 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 PhosphoPRAS40 (Thr246) while Total PRAS40 levels remained unchanged.
Validation of Phospho (Thr246)/total PRAS40 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.
Differential expression of PRAS40 Total in various human and mouse cell lines
Various human and mouse cell lines were seeded at 40,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO2. The cells were lysed the following day with 100 µL of Lysis Buffer.
PRAS40 Total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL (approximately 4,000 cells) of cell lysate were 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.
Total PRAS40 expression was detected across all human and mouse cell lines tested, demonstrating assay versatility.
