AlphaLISA SureFire Biotin-Free Human and Mouse Phospho-Rb (Thr826) Detection Kit, 500 Assay Points

Phospho-AlphaLISA SureFire Biotin Free 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 Biotin Free 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 Biotin Free one-plate assay protocol

Detection of Phosphorylated target protein with AlphaLISA SureFire Biotin Free 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 quality.

Decrease of Rb Phospho (Thr826) in Palbociclib treated cells

THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in complete RPMI 1640 medium and treated with increasing concentrations of Palbociclib for 24 hours at 37°C, 5% CO₂.

After treatment, the cells were lysed with the addition of 50 µL 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Rb Phospho (Thr826) and Rb Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, lysate was further diluted 1:2 with Lysis Buffer,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, Palbociclib triggered a dose-dependent decrease in the levels of Phospho (Thr826) and Total Rb.

Decrease of Rb Phospho (Thr826) in CDK6 PROTAC treated cells

THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in complete RPMI 1640 medium and treated with increasing concentrations of BSJ-03-123 (CDK6 PROTAC) for 24 hour at 37°C, 5% CO₂.

After treatment, the cells were lysed with the addition of 50 µL 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Rb Phospho (Thr826) and ERK1/2 Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, lysate was further diluted 1:2 with Lysis Buffer, 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, BSJ-03-123 induced a significant decrease in the levels of Rb Phospho (Thr826) while Total ERK1/2 levels remain unchanged.