Description
Overview Mixed lineage kinase domain-like protein is encoded by the MLKL gene in murine species. MLKL is a pseudokinase that plays a crucial role in TNF-induced necroptosis. MLKL is activated following phosphorylation by RIP3 kinase, which induces a conformational switch essential for necroptosis. The protein kinase domain of MLKL is catalytically inactive, but contains a pseudoactive site with an interaction between Lys-219 and Gln-343 residues. Upon phosphorylation by RIP3, MLKL undergoes an active conformation and homodimerization through its amino-terminal coiled-coil domain. The AlphaLISA™ SureFire® Ultra ™ mouse p-MLKL (Ser345) assay is a sandwich immunoassay for quantitative detection of Phospho-MLKL (Ser345) 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.
