AlphaLISA SureFire Ultra Human Phospho-STAT4 (Tyr693) 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.

Activation of STAT4 Phospho (Tyr693) in NK-92 cells

NK-92 cells were seeded in a T175 flask at 500K/mL in complete MEMα medium deprived of IL-2 and incubated overnight. Cells were resuspended in HBSS + 0.1% BSA, seeded in a 96-well plate (500,000 cells/well) and treated with increasing concentrations of IL-12 for 30 minutes.

After treatment, the cells were lysed with the addition of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT4 Phospho (Tyr693) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 20,000 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, IL-12 triggered a dose-dependent increase in the levels of STAT4 Phospho (Tyr693) while Total levels remained unchanged.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from NK-92 cells IL-2 starved for 18 hours and stimulated with 200 ng/mL IL-12 for 30 minutes in HBSS + 0.1% BSA. Cells were lysed at 3.2 x 10⁶ with the addition of 5X Lysis Buffer for 10 minutes at RT with shaking.

Lysates were serially diluted in Lysis Buffer and assayed for Phospho (Tyr693) and Total STAT4 levels using respective AlphaLISA SureFire Ultra kits. 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. The assay can detect STAT4 Phospho (Tyr693) in less than 500 cells/datapoint.