AlphaLISA SureFire Ultra Human IRF5 Aggregate 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.

Increase of IRF5 Aggregate in CpG-B ODN-2006 treated cells

RPMI 8226 cells were seeded in a 96-well plate (500,000 cells/well) in complete medium and incubated for approximately 10 minutes at 37°C, 5% CO₂. The cells were then treated with increasing concentrations of CpG-B ODN 2006 for 3 hours.

After treatment, the cells were spun down at 1200 rpm for 5 minutes, washed with HBSS + 0.1% BSA and lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Aggregate, Phospho (Ser446) and Total IRF5 levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 50,000 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, CpG-B ODN 2006 triggered a dose-dependent increase in the levels of Aggregate and Phospho (Ser446) IRF5 while Total IRF5 levels remained unchanged.

Increase of IRF5 Aggregate in Calyculin A treated cells

THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium and incubated for approximately 10 minutes at 37°C, 5% CO₂. The cells were treated with increasing concentrations of Calyculin A for 3 hours.

After treatment, the cells were spun down at 1200 rpm for 5 minutes, washed with HBSS + 0.1% BSA and lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Aggregate, Phospho (Ser446) and Total IRF5 levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 50,000 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, Calyculin A triggered a dose-dependent increase in the levels of Aggregate and Phospho (Ser446) IRF5 while Total IRF5 levels remained unchanged.