Description
Overview
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
How it works
Phospho-AlphaLISA
SureFire Ultra
assay principle
The Phospho-AlphaLISA
SureFire Ultra
assay measures a protein target when phosphorylated at a specific residue.
The assay uses two antibodies which recognize the phospho epitope and a distal epitope on the targeted 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 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 permits the cells 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 AlphaLISA
SureFire Ultra
quality.
