IRAK4 (IL-1R-associated kinase 4) is an essential serine-threonine kinase in the signaling cascade associated with toll-like receptors (TLRs). Through NF-κB-mediated and mitogen-activated protein kinase (MAPK) activation as well as interferon response factors 3 and 7 (IRF3/7), IRAK4 is implicated in the generation of proinflammatory cytokines. IRAK4 is a key player in the IL-1/TLR signaling pathway because, unlike other IRAK proteins, it fully inhibits TLR receptor activation when it is suppressed. IRAK4 is an important drug therapy target for various cancers and inflammatory disorders including rheumatoid arthritis, inflammatory bowel disease, and other autoimmune diseases.
The AlphaLISA SureFire Human Phospho-IRAK4 (Thr345/Ser346) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-IRAK4 (Thr345/Ser346) 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
Alpha SureFire kits can be used for
Cellular kinase assays
Receptor activation studies
Screening
Phospho-AlphaLISA SureFire Ultra assay principle
The Phospho-AlphaLISA SureFire Ultra assay measures a target protein when phosphorylated at a specific residue in a biological sample (e.g. cell lysate).
The assay uses two antibodies which recognize the phospho epitope and a distal epitope on the 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 assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of phosphorylated 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 phosphoprotein present in the sample.
Phospho-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 Phospho-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.
Phospho-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of Phosphorylated 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 robust AlphaLISA SureFire Ultra quality.
LPS induction of Phospho IRAK4 (Thr345/Ser346) in primary macrophages
Monocytes were isolated from PBMC fractions extracted from healthy donors and cultured for 6 days in complete DMEM containing 20 ng/mL M-CSF to differentiate them into macrophages. Macrophages were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated for overnight at 37°C, 5% CO2 . Cells were treated with 1 µg/mL LPS for 30 minutes.
After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRAK4 Phospho (Thr345/Ser346) levels were evaluated using the AlphaLISA SureFire Ultra assay. 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.
As expected, LPS triggered an increase in the levels of Phospho (Thr345/Ser346) IRAK4 in primary macrophages.
IL-1β induction of Phospho IRAK4 (Thr345/Ser346)
Karpas 299 cells were washed, resuspended in HBSS + 0.1% BSA at a density of 3 x 106 cells/mL and seeded in a 96-well plate (300,000 cells/well). Cells were treated with increasing concentrations of IL-1β 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 Phospho (Thr345/Ser346) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 12,000 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, IL1-β triggered a dose-dependent increase in the levels of Phospho (Thr345/Ser346) IRAK4 with a small decrease in Total levels.
LPS induction of Phospho IRAK4 in mouse macrophages
RAW264.7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated for 48 hours at 37°C, 5% CO2. Cells were treated with increasing concentrations of LPS for 15 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRAK4 Phospho (Thr345/Ser346) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. 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.
As expected, LPS triggered a dose-dependent increase in the levels of Phospho (Thr345/Ser346) IRAK4 while Total levels were unchanged.
