AlphaLISA SureFire Ultra Human and Mouse Total GSPT1 Detection Kit, 100 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.

Time-dependent GSPT1 degradation in MOLT-4 cells

MOLT-4 cells were seeded in a 96-well plate (250,000 cells/well) in HBSS + 0.1% BSA. The cells were treated with increasing concentrations of CC-885 for up to 6 hours.

After treatment, the cells were lysed with 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). GSPT1 levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 10,000 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.

The DC50 decreases with the length of CC-885 treatment, demonstrating an increase of potency over time.

Specific degradation of GSPT1 using CC-885 PROTAC

MOLT-4 cells were seeded in a 96-well plate (250,000 cells/well) in HBSS + 0.1% BSA. The cells were treated for 1 hour with 5 µM MG132, followed by increasing concentrations of CC-885 for 2 hours.

After treatment, the cells were lysed with 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). GSPT1 levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 10,000 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, CC-885 induced a dose-dependent decrease in GSPT1 Total levels. The lack of protein degradation in the presence of MG132 confirmed that the degradation was driven by the ubiquitin-proteosome system.

Ramos cells were seeded in a 96-well plate (100,000 cells/well) in complete medium. The cells were treated with increasing concentrations of GBD-9 for 16 hours.

After treatment, the cells were spun down, washed with HBSS and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). GSPT1 and Cofilin Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 10,000 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, GBD-9 treatment resulted in a dose-dependent decrease in the levels of Total GSPT1.

MOLT-4 cells were seeded in a 96-well plate (250,000 cells/well) in complete medium. The cells were treated with increasing concentrations of Eragidomide or SJPYT-195 for 16 hours.

After treatment, the cells were spun down, washed with HBSS and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). GSPT1 and Cofilin Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 25,000 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, treatment with Eragidomide or SJPYT-195 resulted in a dose-dependent decrease in the total levels of GSPT1.

Selectivity of GSPT1 Total assay

Selectivity of the Total GSPT1 assay was assessed by assaying recombinant GSPT1 protein.

Dilutions of recombinant GSPT1 protein (Abcam ab12287) was prepared in Lysis Buffer and evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of protein 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.

Expression levels of GSPT1 in a variety of cell types

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 for 10 minutes at RT with shaking (350 rpm). Suspension cells were seeded at 100,000 cells/well (200 µL/well) in a 96-well culture plate in HBSS + 0.1% BSA and lysed with 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm).

Total GSPT1 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (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.

GSPT1 is highly expressed in most cell lines tested.