IRAK1 homodimers represent an active signaling state that forms during TLR/IL-1R pathway activation and contributes to signal amplification. Following IRAK4-mediated phosphorylation, IRAK1 molecules undergo trans-autophosphorylation and form dimeric complexes with enhanced kinase activity. The dimeric form shows increased catalytic efficiency and enhanced ability to recruit TRAF6, amplifying the inflammatory signal. Hyperactive IRAK1 dimerization contributes to excessive inflammation in autoimmune diseases and supports oncogenic signaling in MYD88-mutant cancers. The dimer interface represents a potential therapeutic target for selective IRAK1 inhibition.
The AlphaLISA SureFire Ultra Human IRAK1 Dimer is a sandwich immunoassay for the quantitative detection of IRAK1 dimer 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 dimer / aggregate detection assay principle
The AlphaLISA SureFire Ultra dimer/aggregate assay measures the levels of a given protein in a dimeric, multimeric or any other aggregated form in a biological sample (e.g. cell lysate).
The assay uses a single antibody clone tagged with both CaptSure™ peptide and biotin, that recognizes exposed epitopes on the multivalent target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure agent to specifically immobilize the antibody labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture the same specific antibody, which is biotinylated. In the presence of the multivalent protein, 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 multimeric protein present in the sample.
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 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 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.
IRAK1 Dimer 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 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.
