AlphaLISA SureFire Ultra Human and Mouse Total IRAK4 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.

Validation of IRAK4 assay in IL-1β treated cells

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, 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 and a small decrease in Total levels.

Validation of IRAK4 assay in LPS treated cells

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.

Versatility of Total IRAK4 assay in various cell lines

Adherent cells were seeded at 50,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO2. Cells were lysed with 50 µL of Lysis Buffer.

Suspension cells were seeded at 50,000 cells/well in a 96-well culture plate in HBSS + 0.1% BSA and lysed with 50 µL of Lysis Buffer. IRAK4 Total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 10,000 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.

IRAK4 is expressed across a wide range of common cell lines.