AlphaLISA SureFire Ultra Human Total IRAK1 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.

Karpas 299 cells were harvested and seeded in a 96-well plate (200,000 cells/well) in DMEM medium containing 10% FBS. The cells were treated with increasing concentrations of JNJ-1013 (IRAK1 PROTAC) for 18 hours. 

After treatment, the cells were spun down and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRAK1 and IRAK4 Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 20,000 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, treatment with JNJ-1013 resulted in a dose-dependent-decrease of IRAK1 Total level while IRAK4 levels remained unchanged.

HEK293T cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of JNJ-1013 (IRAK1 PROTAC) for 18 hours. 

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRAK1 and IRAK4 Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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, treatment with JNJ-1013 resulted in a dose dependent-decrease of IRAK1 Total level while IRAK4 levels remained unchanged.

Knockout validation of IRAK1 Total assay

IRAK1 Total levels were assessed in HAP1 wild type (WT) and IRAK1 knockout (KO) cells. Cells were cultured to confluency in T175 flasks at 37°C, 5% CO₂ and then lysed in 4 mL of Lysis Buffer.

Lysates were serially diluted in Lysis Buffer and IRAK1 Total 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.

IRAK1 Total was only detected in WT cells, confirming assay specificity.

Expression of IRAK1 in various cell lines

Adherent cell lines were seeded in a 96-well plate (40,000 cells/well) and incubated overnight at 37°C, 5% CO₂. Cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm).

Suspension cell lines were seeded in a 96-well plate (400,000 cells/well) and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm).

IRAK1 levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 4,000 adherent cells and 40,000 suspension 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, Total IRAK1 protein is expressed in a wide range of cell types.

Sensitivity of the Total IRAK1 assay was assessed by assaying recombinant IRAK1 protein (ab268680).

Dilutions of recombinant IRAK1 protein were prepared in Lysis Buffer and evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of protein 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.

Cell lysate was prepared from HEK293T cells cultured to confluency in a T175 flask and lysed in 4 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and IRAK1 Total levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate 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.

Approximate number of cells per datapoint is indicated. The dotted line represents assay background. This assay can detect IRAK1 expression in less than 100 cells/datapoint.