AlphaLISA SureFire Ultra Human Phospho-STAT1 (Tyr701)/phospho-STAT2 (Tyr690) Complex Detection Kit, 10,000 Assay Points

AlphaLISA SureFire Ultra phospho complex detection 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 complex AlphaLISA SureFire Ultra detection reagents. This protocol allows cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

AlphaLISA SureFire Ultra phospho protein complex detection one-plate assay protocol

Detection of the phospho protein complex 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 enables miniaturization while maintaining robust AlphaLISA SureFire Ultra quality.

A431 cells were seeded in a 96-well plate (60,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of IFNβ for 30 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT1 Phospho (Tyr701)/STAT2 Phospho (Tyr690) Complex and STAT2 Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 6,000 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, IFNβ triggered a dose-dependent increase in the levels of p-STAT1/p-STAT2 Complex with a modest decrease in Total STAT2 levels (2.8 fold).

Decrease of STAT1/STAT2 Phospho Complex by Ruxolitnib

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

After treatment, the cells were spun down at 1200 rpm for 5 minutes and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT1 Phospho (Tyr701)/STAT2 Phospho (Tyr690) Complex and Total STAT2 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 decrease of phosphorylated STAT1/STAT2 Complex 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 STAT1 Phospho (Tyr701)/STAT2 Phospho (Tyr690) Complex 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 p-STAT1/p-STAT2 complex in less than 1,000 cells.