AlphaLISA SureFire Ultra Human and Mouse Total PDGFRα Detection Kit, 500 Assay Points

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 permits the cells 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 AlphaLISA SureFire Ultra quality.

PDGF Receptor α activation in cells treated with PDGF-BB

NIH/3T3 cells were seeded in a 96-well plate (50,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of PDGF-BB for 10 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). PDGF Receptor α Phospho (Tyr720) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 5,000 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, the PDGF-BB triggered a dose-dependent increase in the levels of Phospho (Tyr720) PDGF Receptor α while Total levels remained unchanged.

PDGF Receptor α inhibition in cells treated with Dasatinib

Caco-2 cells were seeded in a 96-well plate (50,000 cells/well) in medium containing 20% FBS and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of Dasatinib for 30 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). PDGF Receptor α Phospho (Tyr720) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 5,000 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, the Dasatinib triggered a dose-dependent decrease in the levels of Phospho (Tyr720) PDGF Receptor α while Total levels remained unchanged.

Knockout validation of PDGF Receptor α Total assay

PDGF Receptor α Total levels were assessed in Wild Type (WT) and PDGFR α knockout (KO) SH-SY5Y (Abcam, ab275335) cells. PDGFR α KO cells and SH-SY5Y WT cells were seeded at various densities in a 96 well plate in complete medium, and incubated overnight at 37°C, 5% CO2.

The cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). PDGFR α levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate 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, PDGF Receptor α was only detected in WT cells demonstrating assay selectivity.

Expression of PDGF Receptor α in various cell lines

Adherent cells were seeded at 40,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO2. Cells were lysed with 100 µL of Lysis Buffer.

Suspension cells were seeded at 400,000 cells/well in a 96-well culture plate in HBSS + 0.1% BSA, cells were spun down and lysed with 100 µL of Lysis Buffer.

PDGF Receptor α Total levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 4,000 adherent cells or 40,000 suspension 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.

PDGF Receptor α expression is dependent upon cell type. High levels of expression were detected in Caco-2 and NIH/3T3 cells.