A solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). A monoclonal capture antibody specific for the insulin receptor (IR) β subtype (regardless of phosphorylation state) has been coated onto the multiwell strips provided with the kit. Standard dilutions and samples are incubated for 2 hours at RT. IR antigen binds to the capture antibody. After a wash, a detection antibody specific for the non-phosphorylated or phosphorylated protein is incubated for 1 hr at RT, which results in binding to the immobilized IR protein. An anti-rabbit IgG-HRP binds to the immobilized protein, completing the four-member sandwich. The reaction is visualized by tetramethylbenzidine (TMB) substrate, followed by the stop solution. The intensity of the yellow color, measured in a multiwell plate reader at 450 nm, is directly proportional to the concentration of IR in the original sample. The unknown concentrations are calculated from the standard curve run with each assay.
The assay is designed for the quantitative determination of human, mouse, and rat IR in cell lysates.
phospho-Insulin Receptor β Subunit (pTyr1162/1163) ELISA is specific for IR β phosphorylated on tyrosines 1162 and 1163.
Insulin receptor (IR) belongs to the superfamily of the growth factor receptor tyrosine kinases that regulate multiple signaling pathways through activation of a series of phosphorylation cascades. Once activated, the IR initiates a variety of metabolic functions including glucose transport, glycogen synthesis, protein synthesis, translational control, and mitogenesis.
Features and Benefits
The IR ELISAs offer a fast (total time 4 hours), sensitive, and specific alternative to immunoblotting and other bioassays.
Sensitivity is <0.8 units/mL, which is 2x more sensitive than immunoblotting. The specificity was confirmed by peptide competition. Only the phosphopeptide containing the phosphorylated tyrosines blocks the ELISA signal.