AlphaLISA SureFire Ultra Human Phospho-Ret (Tyr905) 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.

GDNF activation of Ret Phospho (Tyr905)

MCF7 cells were seeded in a 96-well plate (40,000 cells/well) in complete media, and incubated overnight at 37°C, 5% CO2. The cells were then starved overnight in serum-free media and treated with increasing concentrations of GDNF for 15 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Ret Phospho (Tyr905) 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, GDNF triggered a dose-dependent increase in the levels of Ret Phospho (Tyr905) while Ret Total levels remained unchanged.

Activation of Ret Phospho (Tyr905) in cells treated with pervanadate

SH-SY5Y cells were seeded in a 96-well plate (40,000 cells/well) and incubated overnight at 37°C, 5% CO2. The cells were treated with 12.5 µM Retinoic Acid for 18 hours and then increasing concentrations of pervanadate for 30 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Ret Phospho (Tyr905) 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, pervanadate caused a dose-dependent increase in the levels of Ret Phospho (Tyr905) while Ret Total levels remained unchanged.