The BioSource Immunoassay Kit AKT [pT308] was designed to detect and quantify the level of AKT protein phosphorylated at threonine residue 308. The detection of phosphorylated AKT is widely used in medical research because activated AKT acts as a key mediator of signals for cell survival, proliferation, angiogenesis, and a number of metabolic effects of insulin. Protein kinase B/AKT and its upstream signal transducer, phosphatidylinositol-3 kinase, play an essential role in the control of transcription and translation. AKT is one of the central players in tumorigenesis and the effects of AKT activation may be mediated by modulation of expression or activity of various molecules including Bcl-2, BAD, caspase-9, eNOS, glycogen synthase and transcription factors.
We have been using the BioSource Immunoassay Kit AKT*[pT308] in our laboratory for the last four months because our research interest is related to the study of the effects of insulin-like growth factor-1 (IGF-1) and its binding proteins on cell proliferation and apoptosis. Although the AKT*[pT308] ELISA kit was designed primarily for human cell lines, there is enough cross-reactivity that we were able to successfully use it with rat mesangial cells which we obtained from ATCC (Manassas, VA, USA). For normalizing the AKT content of samples we used the AKT (Total) ELISA kit (also from BioSource).
The protocol of the AKT*[pT308] ELISA kit is straightforward and takes about 5 hours (including incubation times). Previously, for the same purpose we used a Western blotting method which, for the same number of samples, was much more time consuming. The principle behind the kit is a solid phase sandwich ELISA. A monoclonal antibody specific for AKT is coated onto the wells of the provided microtiter strips and samples are then pipetted into these wells. During the first incubation, the AKT antigen binds to the immobilized antibody. After washing, rabbit antibody specific for AKT phosphorylated at threonine 308 is added to the wells. During the second incubation this antibody serves as a detector by binding to the immobilized AKT protein captured during the first incubation. After removal of excess detection antibody an anti-rabbit IgG-HRP is added. Following a third incubation and washing a substrate solution 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 AKT [pT308] present in the original specimen.
As with other ELISAs some additional equipment is needed: a microtiter plate reader capable of measurement at or near 450 nm, a plate washer, graph paper and other supplies. In addition it is a good idea to purchase, or prepare ahead of time, the protease inhibitor cocktail (we buy ours from Sigma) and Cell Extraction Buffer (from BioSource). For quantification analysis it is necessary to have the AKT [pT308] Standard, which will take 8 out of 96 wells of your control and experimental samples. Keep in mind that the standard should be used within 1 hour of reconstitution. The kit is relatively expensive but compensates by saving time and effort.
The AKT*[pT308] ELISA kit is an appropriate choice for researchers who study cell signal pathways. The accuracy and convenience of the kit make it a great laboratory asset. We are pleased with this product and would recommend it to other laboratories.
Tetyana L. Vasylyeva, M.D., Ph.D.
Postdoctoral Fellow
Endocrine Division , Department of Pediatrics
University of Texas Health Science Center at San Antonio