The IRAK1/IRAK4 complex is a critical signaling module in TLR and IL-1R pathways that mediates innate immune responses to pathogens. Upon receptor activation, MYD88 recruits IRAK4, which then recruits and phosphorylates IRAK1, forming an active kinase complex. IRAK4 phosphorylates IRAK1 at multiple sites, triggering autophosphorylation and full activation. The activated complex recruits TRAF6, leading to NF-κB and MAPK pathway engagement. Dual IRAK1/IRAK4 inhibitors are being developed as anti-inflammatory therapeutics for autoimmune diseases and MYD88-mutant lymphomas.
The AlphaLISA SureFire Ultra Human IRAK1 / IRAK4 Complex is a sandwich immunoassay for the quantitative detection of IRAK1 / IRAK4 complex in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
AlphaLISA SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
AlphaLISA SureFire Ultra protein complex detection assay principle
The AlphaLISA SureFire Ultra complex assay measures cellular protein-protein interactions (PPI) in a biological sample (e.g. cell lysate).
The assay uses two antibodies which recognize Protein 1 and Protein 2, respectively. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the first assay antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture the second antibody, which is biotinylated. In the presence of the protein complex, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of PPI present in the sample.
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 enables cell 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.
IRAK1/IRAK4 Complex is induced upon IL-1b stimulation
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.
