Invitrogen™ Human AKT (Phospho) [pS473] ELISA Kit

ELISA

The Human AKT (Phospho) [pS473] ELISA Kit is designed to detect and quantify the level of AKT (Phospho) [pS473] in fresh or frozen human cell lysates. The assay recognizes both natural and recombinant AKT (Phospho) [pS473]. Principle of the method A monoclonal capture antibody specific for AKT (Phospho) [pS473] has been coated onto the wells of the 96-well plate. During the first incubation, standards of known content and unknown samples are pipetted into the wells and the antigen binds to the immobilized (capture) antibody. After washing, a rabbit antibody specific for the target protein is added to the wells and serves as a detection antibody by binding to the immobilized protein captured during the first incubation. After washing, a horseradish peroxidase labeled anti-rabbit IgG is added. This binds to the detection antibody to complete the four member sandwich. After a third incubation and washing to remove all the unbound enzyme, a substrate solution (TMB) is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of target protein present in the original specimen and the optical density can be read on a standard microplate reader. Rigorous validation Each manufactured lot of this ELISA kit is quality tested for criteria such as sensitivity, specificity, precision, and lot-to-lot consistency. See manual for more information on validation.