AlphaLISA SureFire Ultra Human and Mouse Total STAT2 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 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.

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 for 15 minutes at 37°C, 5% CO₂. The cells were then treated with increasing concentrations of JAK inhibitor, Ruxolitinib for a further 1 hour.

After treatment, the cells were spun at 1200 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 decrease of phosphorylated STAT2 after 1 hour while STAT2 Total levels remained unchanged.

Evaluation of STAT2 Total levels in PBMCs

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from healthy donors using Ficoll Plaque Plus (Merck GE17-1440-02). PBMCs were lysed at 6 x 10⁶ cells/mL with Lysis Buffer for 10 minutes at RT with shaking. Lysate was serially diluted in Lysis Buffer and evaluated for STAT2 Total levels using the AlphaLISA SureFire Ultra kit.

For the detection step, 10 µL of cell lysate (starting at approximately 60,000 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. Dotted line represents assay background.

Expression of STAT2 in various cell lines

Adherent cell lines were seeded in a 96-well plate (40,000 cells/well) and incubated overnight at 37°C, 5% CO2. Cells were lysed with 100 µL of Lysis Buffer at RT with shaking (350 rpm). Suspension cell lines were seeded in a 96-well plate (400,000 cells/well) in HBSS + 0.1% BSA and then lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm).

STAT2 levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 4,000 adherent cells and 40,000 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.

STAT2 is expressed in a wide variety of cell lines. High levels of expression were detected in PC3, A431 and RPMI 8226 cells.

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.

Lysate was serially diluted in Lysis Buffer and STAT2 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 Total STAT2 in less than 300 cells.