The IRAK4/MYD88 complex serves as the primary signaling platform that initiates TLR and IL-1R signaling cascades essential for innate immune activation. Upon receptor engagement, MYD88 oligomerizes to form the "myddosome," which recruits IRAK4 through death domain interactions. IRAK4 binding promotes trans-autophosphorylation and kinase activation, then recruits and phosphorylates IRAK1 and IRAK2. Oncogenic MYD88 L265P mutations create hyperactive IRAK4/MYD88 complexes that drive constitutive NF-κB signaling in B-cell malignancies. Targeting the IRAK4/MYD88 interaction represents a promising therapeutic approach for autoinflammatory diseases and MYD88-mutant cancers.
The AlphaLISA SureFire Ultra Human IRAK4 / MyD88 Complex is a sandwich immunoassay for the quantitative detection of IRAK4 / MyD88 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.
IRAK4/MyD88 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). IRAK4/MyD88 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 IRAK4/MyD88 Complex 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). IRAK4/MyD88 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 IRAK4/MyD88 Complex within 5 minutes, peaking after 60 minutes of treatment.
IL-1b induces IRAK4/MyD88 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 the indicated concentrations of IL-1b for 10 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). IRAK4/MyD88 Complex, Total IRAK4 and MyD88 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, IRAK4/MyD88 Complex formation was upregulated in a dose-dependent manner, while a modest decrease was observed in IRAK4 Total levels. No changes were observed in Total MyD88 levels.
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 IRAK4/MyD88 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 IRAK4/MyD88 Complex down to 500 cells/datapoint.
