AlphaLISA SureFire Ultra Human Phospho-IRF5 (Ser446) Detection Kit, 100 Assay Points

Phospho-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 Phospho-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.

Phospho-AlphaLISA SureFire Ultra one-plate assay protocol

Detection of Phosphorylated 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.

Activation of Phospho IRF5 (Ser446) in R848 treated cells

RPMI 8226 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 1 µM of TLR7/8 agonist, R848 (MedChem Express HY-13740) at the indicated timepoints.

After treatment, the cells were spun down at 1200 rpm for 5 minutes, washed with HBSS + 0.1 % BSA and then lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRF5 Phospho (Ser446) was evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 20,000 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.

R848 triggered a modest increase in Phospho (Ser446) IRF5, which peaked at 6 hours.

Increase of Phospho IRF5 (Ser446) in Calyculin A treated cells

THP-1 cells were seeded in a 96-well plate (40,000 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 2.5 hours.

After treatment, the cells were spun down at 1200 rpm for 5 minutes, washed with HBSS + 0.1 % BSA and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRF5 Phospho (Ser446) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. 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, Calyculin A triggered a dose-dependent increase in the levels IRF5 Phospho (Ser446) while Total IRF5 levels remained unchanged.