AlphaLISA SureFire Ultra Human and Mouse Phospho-PDGFRα (Tyr720) Detection Kit, 10,000 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.

Phosphorylation of PDGF Receptor α 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.

Phosphorylation of PDGF Receptor α in cells treated with PDGF-BB

Caco-2 cells were seeded in a 96-well plate (40,000 cells/well) in medium containing 20% FBS 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 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, the PDGF-BB triggered a dose-dependent increase in the levels of Phospho (Tyr720) PDGF Receptor α while Total levels remained unchanged.

Phosphorylation of PDGF Receptor α in cells treated with PDGF-AA

Caco-2 cells were seeded in a 96-well plate (40,000 cells/well) in medium containing 20% FBS and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of PDGF-AA 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 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, the PDGF-AA triggered a modest and 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, Dasatinib triggered a dose-dependent decrease in the levels of Phospho (Tyr720) PDGF Receptor α while Total levels remained unchanged.

PDGF Receptor α inhibition in cells treated with Imatinib

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 Imatinib for 6 hours.

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, Imatinib 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 Phospho (Tyr720) assay

PDGF Receptor α Phospho (Tyr720) 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 treated with 2.5 µM Pervanadate 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 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 Phospho (Tyr720) was only detected in the pervanadate treated WT cells.