AlphaLISA SureFire Ultra Human and Mouse Phospho-RAB10 (Thr73) Detection Kit, 50,000 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.

RAW 264.7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated for 48 hours at 37°C, 5% CO₂. The cells were treated with increasing concentrations of chloroquine for 60 minutes.

After treatment, the cells were lysed with 100 µL Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Thr73) and Total RAB10 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 8,000 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, chloroquine treatment leads to increased RAB10 phosphorylation with no significant changes in Total RAB10 levels.

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 chloroquine for 5 hours.

After treatment, the cells were lysed with 100 µL Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Thr73) and Total RAB10 levels were evaluated by AlphaLISA SureFire Ultra. 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, chloroquine treatment leads to increased RAB10 phosphorylation with no change in Total RAB10 levels.

Inhibition of RAB10 phosphorylation

RAW 264.7 cells were seeded in a 96-well plate (80,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of MLi-2 in the presence of 50 ng/mL chloroquine for 3 hours.

After treatment, the cells were lysed with 100 µL Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Thr73) and Total RAB10 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 8,000 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, MLi-2 treatment resulted in a dose dependent decrease in RAB10 (Thr73) phosphorylation, no decrease in Total RAB10 levels was observed.

RAB10 assay sensitivity - cell lysate dilution

Cell lysate was prepared from A549 cells cultured to confluence in T175 flasks and treated with 250 µg/mL chloroquine for 5 hours. Cells were lysed with 10 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and RAB10 Phospho (Thr73) and Total levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. 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.

Approximate number of cells/datapoint is indicated on the graph. The dotted line represents assay background. This assay can detect RAB10 Phospho (Thr73) in less than 1,000 cells/datapoint.