AlphaLISA SureFire Ultra Mouse Total IRF9 Detection Kit, 500 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.

RAW 264.7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of Ruxolitinib for 8 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Total IRF9 and Cofilin levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells for IRF9 and 400 cells 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.

Inhibition of JAK1 signaling, mediated by Ruxolitinib induced a significant decrease of Total IRF9 levels after 8 hours, while Cofilin Total levels remained unchanged.

RAW 264.7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO₂. The cells were treated 5 µM of Pacritinib for 8 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Total IRF9 and Cofilin levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells for IRF9 and 400 cells 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.

Inhibition of JAK1 signaling, mediated by Pacritinib, induced a significant decrease of Total IRF9 levels after 8 hours, while Cofilin Total levels remained unchanged (data not shown).

Total IRF9 expression in mouse 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).

IRF9 levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 4,000 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.

As expected, basal Total IRF9 expression is low in most mouse cell lines tested. High levels of expression were detected in MEF cells and moderate expression in RAW 264.7 cells.