AlphaLISA SureFire Ultra Human Total STAT5 Detection Kit, 500 Assay Points

Total-AlphaLISA SureFire Ultra assay principle

The Total-AlphaLISA SureFire Ultra assay measures the expression level of a protein target in a cell lysate.

The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the targeted protein. AlphaLISA assays require two bead types: Acceptor and Donor beads. Acceptor beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure™ tag. Donor beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of targeted protein, the two antibodies bring the Donor and Acceptor beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor bead, allowing the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.

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.

IFNα and IFNγ induce STAT5 phosphorylation in a dose-dependent manner

PBMCs were isolated from healthy donors and cultured for 6 days in complete DMEM containing 20 ng/mL M-CSF to differentiate them into macrophages. Macrophages were seeded in a 96-well plate (40,000 cells/well) in complete DMEM, and incubated overnight at 37°C, 5% CO2. Cells were starved for 2 hours and then treated with IFNα or IFNγ for 20 minutes.

After treatment, cells were lysed in 150 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT5 Phospho (Tyr694/699) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,600 cells) were 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.

As expected, IFNα and IFNγ triggered a dose-dependent increase in the levels of Phospho STAT5 (Tyr694/699) while Total STAT5 levels remained unchanged.

Degradation of Total STAT5 in PROTAC treated cells

MOLT-4 cells were seeded in a 96-well plate (200,000 cells/well) in medium containing 1% FBS and treated with increasing concentrations of STAT5 PROTAC, AK-2292 for 20 hours at 37°C, 5% CO2.

After treatment, the cells were spun at 1200 rpm for 5 minutes, washed with HBSS + 0.1 % BSA and then lysed with 100 µL of Lysis Buffer for 10 minutes at RT with sharking (350 rpm). STAT5 Total and STAT3 Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 8,000 cells) were 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, STAT5 PROTAC AK-2292 triggered a dose-dependent decrease in the levels of Total STAT5 while Total STAT3 levels remained unchanged.