The Human AKT (Total) ELISA Kit is a solid-phase sandwich Enzyme-Linked Immunosorbent Assay (ELISA) designed to detect and quantify the level of AKT (Total) in fresh or frozen human cell lysates. Cross-reactivity in mouse and rat cell lysates has been observed. The assay recognizes both natural and recombinant AKT (Total), independent of its phosphorylation state.
Principle of the method
A monoclonal capture antibody specific for AKT (Total) 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.
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.
AKT (protein kinase B, PKB, RAS-alpha) is a 57kDa serine/threonine kinase that plays an important role in diverse biological responses such as regulation of metabolism, cell survival and growth by phosphorylating multiple proteins. AKT is activated by insulin, PI3K, IGF1 and various other growth and survival factors. AKT promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including forkhead transcription factors, and caspase-9. There are three mammalian isoforms of Akt: AKT1 (PKB alpha), AKT2 (PKB beta) and AKT3 (PKB gamma) with AKT2 and AKT3 being approximately 82% identical with the AKT1 isoform. Each isoform has a pleckstrin homology (PH) domain, a kinase domain and a carboxy terminal regulatory domain. AKT was originally cloned from the retrovirus AKT8, and is a key regulator of many signal transduction pathways. The AKT pathway is a major target for cancer therpeutics where AKT signaling dysfunction has implicated in many types of cancer, including cancer syndromes known as phakomatoses.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.