AlphaLISA SureFire Ultra Human and Mouse Phospho-IRAK4 (Thr345/Ser346) 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.

LPS induction of Phospho IRAK4 (Thr345/Ser346) in primary macrophages

Monocytes were isolated from PBMC fractions extracted from healthy donors and cultured for 6 days in complete DMEM containing 20 ng/mL M-CSF to differentiate them into macrophages. Macrophages were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated for overnight at 37°C, 5% CO2 . Cells were treated with 1 µg/mL LPS for 30 minutes.

After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRAK4 Phospho (Thr345/Ser346) levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 8,000 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, LPS triggered an increase in the levels of Phospho (Thr345/Ser346) IRAK4 in primary macrophages.

IL-1β induction of Phospho IRAK4 (Thr345/Ser346)

Karpas 299 cells were washed, resuspended in HBSS + 0.1% BSA at a density of 3 x 10⁶ cells/mL and seeded in a 96-well plate (300,000 cells/well). Cells were treated with increasing concentrations of IL-1β for 10 minutes.

After treatment, the cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRAK4 Phospho (Thr345/Ser346) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 12,000 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, IL1-β triggered a dose-dependent increase in the levels of Phospho (Thr345/Ser346) IRAK4 with a small decrease in Total levels.

LPS induction of Phospho IRAK4 in mouse macrophages

RAW264.7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated for 48 hours at 37°C, 5% CO2. Cells were treated with increasing concentrations of LPS for 15 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRAK4 Phospho (Thr345/Ser346) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 8,000 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, LPS triggered a dose-dependent increase in the levels of Phospho (Thr345/Ser346) IRAK4 while Total levels were unchanged.