AlphaLISA SureFire Ultra Human STAT2 / IRF9 Complex Detection Kit, 100 Assay Points

AlphaLISA SureFire Ultra 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 complex 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.

AlphaLISA SureFire Ultra protein complex detection one-plate assay protocol

Detection of the 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 allows for miniaturization while maintaining robust AlphaLISA SureFire Ultra quality.

A431 cells were seeded in a 96-well plate (30,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were treated with increasing concentrations of IFNβ for 24 hours.

After treatment, the cells were lysed with 50 µL or 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT2/IRF9 Complex and Cofilin Total levels were evaluated using AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 6,000 cells for Complex and 200 for Cofilin) 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 STAT2/IRF9 Complex formation with no change to Cofilin levels.

THP-1 cells were seeded in a 96-well plate (200,000 cells/well) and treated with 10 ng/mL IFNβ or IFNγ for 24 hours in complete medium at 37°C, 5% CO₂.

After treatment, the cells were washed with HBSS and then lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT2/IRF9 Complex levels were evaluated using the AlphaLISA SureFire Ultra assay. 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, treatment with type I or type II interferons increased STAT2/IRF9 Complex formation with no change to Cofilin Total (data not shown).

RPMI 8226 cells were seeded in a 96-well plate (200,000 cells/well) and treated with 10 ng/mL IFNβ for 24 hours in complete medium at 37°C, 5% CO₂.

After treatment, the cells were washed with HBSS and then lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT2/IRF9 Complex levels were evaluated using the AlphaLISA SureFire Ultra assay. 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, treatment with IFNb increased STAT2/IRF9 Complex formation with no change to Cofilin Total (data not shown).

Assay sensitivity - cell lysate

Cell lysate was prepared from A431 cells cultured to confluence in T175 flasks at 37°C, 5% CO₂. Cells were treated with IFNβ for 24 hours and then lysed in 2 mL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm).

Lysate was serially diluted in Lysis Buffer and assayed for STAT2/IRF9 Complex levels 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 room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour in the dark. The plate was read on an Envision using standard AlphaLISA settings.

Approximate number of cells/datapoint is indicated on the graph. This assay can detect STAT2/IRF9 Complex in less than 3,000 cells. The dotted line represents assay background.