AlphaLISA SureFire Ultra Human IRAK1 Dimer Detection Kit, 500 Assay Points

AlphaLISA SureFire Ultra dimer / aggregate 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 dimer or aggregate AlphaLISA SureFire Ultra detection reagents. This protocol allows for 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 dimer / aggregate detection one-plate assay protocol

Detection of the dimer or aggregate 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 Dimer 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 rapidly induced IRAK1 Dimer levels within just 2 minutes. These levels started to decline after 5 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 Dimer 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 Dimer within just 2 minutes, peaking at 15 minutes of treatment.

IL-1b induces IRAK1 Dimer 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 2 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). IRAK1 Dimer and Total IRAK1 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 Dimer was significantly upregulated in a dose-dependent manner while a 4-fold decrease was observed in IRAK1 Total levels.

IRAK1 PROTAC effects on IRAK1 Dimer

Karpas 299 cells were harvested, washed in DMEM containing 10% FBS and seeded in a 96-well plate (100,000 cells/well). Cells were treated with increasing concentrations of IRAK1 PROTAC JN-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 Dimer 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 5.5-fold decrease of IRAK1 Dimer 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 Dimer 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 Dimer down to 500 cells/datapoint.