AlphaLISA SureFire Ultra Human Total SALL4 Detection Kit, 500 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.

BeWo 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 NVP-DKY709 for 24 hours.

After treatment, the cells were washed in HBSS and lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm) and further diluted before loading. Total SALL4 and Cofilin levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 cells for Total SALL4 and 200 cells for Total Cofilin) 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.

In agreement with literature, NVP-DKY709 degraded around 50% of SALL4 protein while Total Cofilin levels remained unchanged.

BeWo cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of Thalidomide for 24 hours.

After treatment, the cells were washed in HBSS and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm) and further diluted before loading. Total SALL4 and Cofilin levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 1,000 cells for SALL4 and 100 cells for Cofilin) 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, Thalidomide treatment resulted in a dose-dependent decrease in the levels of Total SALL4.

SALL4 expression in various cell lines

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. Suspension cells were washed with HBSS and lysed with Lysis Buffer at 4 x 10⁶ cells/mL.

SALL4 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (4,000 adherent cells or 40,000 suspension 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, SALL4 expression was low or undetectable in most of the cell lines tested. Moderate expression levels were observed in BeWo cells.