AlphaLISA SureFire Ultra Human and Mouse Phospho-STAT2 Tyr690 Detection Kit, 500 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.

Decrease of STAT2 phosphorylation by Ruxolitnib

THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in HBSS containing 10 ng/mL IFNβ at 37°C, 5% CO₂ for 15 minutes. The cells were then treated with increasing concentrations of JAK inhibitor, Ruxolitinib for 1 hour.

After treatment, the cells were spun down at 1,200 rpm for 5 minutes and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT2 Phospho (Tyr690) 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.

Inhibition of JAK1 signaling mediated by Ruxolitinib, resulted in a significant decrease of phosphorylated STAT2 while STAT2 Total levels remained unchanged.

Assay sensitivity – cell lysate dilution

Cell lysate was prepared from A431 cells cultured to confluency in T175 flasks. Cells were treated with 10 ng/mL IFNβ for 30 minutes and then lysed in 4 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and STAT2 Phospho (Tyr690) levels were evaluated using the AlphaLISA SureFire Ultra kit. 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 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 STAT2 Phospho (Tyr690) in less than 200 cells.