Description
Overview The AlphaLISA™ SureFire® Ultra ™ p-JNK1/2/3 (Thr183/Tyr185) assay is a sandwich immunoassay for quantitative detection of phospho-JNK1/2/3 (phosphorylated on Thr183/Tyr185) in cellular lysates using Alpha Technology. In the AlphaLISA™ SureFire® Ultra ™ assay, Donor beads are coated with streptavidin to capture one of the antibodies, which is biotinylated. Acceptor beads are coated with a proprietary CaptSure™ agent that immobilizes the other antibody, labeled with a CaptSure™ tag. In the presence of phosphorylated protein, the two antibodies bring the Donor and Acceptor beads close together, generating signal. The amount of light emission is directly proportional to the amount of phosphoprotein present in the sample. 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.
