AlphaLISA SureFire Ultra Human Total STAT4 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 enables cell 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 robust AlphaLISA SureFire Ultra quality.

Validation of STAT4 Total 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.

Versatility of STAT4 Total assay in various cell lines

Adherent cells were grown to confluency in a T175 flask at 37°C, 5% CO₂, and were lysed with Lysis Buffer at a density of 0.5 x 10⁶ cells/mL. Suspension cells were harvested, washed in HBSS and lysed with Lysis Buffer at 1.6 x 10⁶ cells/mL.

Total STAT4 levels were evaluated using the AlphaLISA SureFire Ultra High Specificity assay. For the detection step, 10 µL of cell lysate (5,000 adherent and 16,000 suspension 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.

STAT4 Total expression was detected highly in NK-92 cells.

STAT4 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 expression in less than 200 cells/datapoint.