AlphaLISA SureFire Ultra Human IRAK1 / IRAK4 Complex Detection Kit, 10,000 Assay Points

AlphaLISA SureFire Ultra complex detection 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 complex AlphaLISA SureFire Ultra detection reagents. This protocol allowing the cells viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

AlphaLISA SureFire Ultra protein complex detection one-plate assay protocol

Detection of the protein complex 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 robust AlphaLISA SureFire Ultra quality.

Karpas 299 cells were harvested, washed in HBSS + 0.1% BSA and seeded in a 96-well plate (400,000 cells/well). Cells were treated with 10 ng/mL IL-1b for the indicated time points.

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). IRAK1/IRAK4 Complex levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 16,000 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.

IL-1b induced IRAK1/IRAK4 Complex formation within 5 minutes and the elevated signal levels persisted for up to 60 minutes.

A549 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO₂. Cells were treated with 10 ng/mL IL-1b for the indicated time points.

After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRAK1/IRAK4 Complex levels were evaluated by AlphaLISA SureFire Ultra. 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.

IL-1b induced IRAK1/IRAK4 Complex formation within 5 minutes, peaking after 60 minutes of treatment.

IL-1b induces IRAK1/IRAK4 Complex in a dose-dependent manner

Karpas 299 cells were harvested, washed in HBSS + 0.1% BSA and seeded in a 96-well plate (400,000 cells/well). Cells were treated with increasing concentrations of IL-1b for 10 minutes.

After treatment, the cells were lysed with the addition of 50 µL 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRAK1/IRAK4 Complex and Total IRAK4 levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 16,000 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, IRAK1/IRAK4 Complex formation was upregulated in a dose-dependent manner, while a modest decrease was observed in IRAK4 Total levels.

IRAK1 PROTAC effects on IRAK1/IRAK4 Complex

Karpas 299 cells were harvested and seeded in a 96-well plate (100,000 cells/well) in DMEM containing 10% FBS. Cells were treated with increasing concentrations of IRAK1 PROTAC JNJ-1013 for 18 hours and then treated with 10 ng/mL IL-1b for 10 minutes.

After treatment, the cells were spun down and lysed with 100 µL Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRAK1/IRAK4 Complex and Total IRAK4 levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 10,000 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.

Treatment with IRAK1 degrader resulted in a 2-fold decrease of IRAK1/IRAK4 Complex levels, while IRAK4 Total levels remained unchanged.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from Karpas 299 cells prepared at 2 x 10⁶ cells/mL and stimulated with 10 ng/mL IL-1b for 10 minutes in HBSS + 0.1% BSA. Cells were lysed with the addition of 5X Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and IRAK1/IRAK4 Complex levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. 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.

Approximate number of cells per datapoint is indicated. The dotted line represents assay background. The assay can detect IRAK1/IRAK4 Complex down to 500 cells/datapoint.