phospho-Akt/PKB (pThr³⁰⁸) ELISA

A solid phase sandwich enzyme-linked immunosorbent assay (ELISA).

The assay is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). A monoclonal capture antibody specific for the Akt/PKB, 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. Akt/PKB 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 Akt/PKB protein. An anti-rabbit IgG-HRP completes 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 Akt/PKB in the original sample. The unknown concentrations are calculated from the standard curve run with each assay.

Akt/PKB ELISAs are designed for in vitro quantitative determination of human, mouse and rat Akt/PKB protein in cell lysates. Akt, also known as protein kinase Bα (PKBα) or RAC-PKa, is one of the downstream targets of PI3 Kinase (PI3K). Akt consist of three highly conserved isoforms designated in humans as Akt1, Akt2, and Akt3. Activated Akt acts as a key mediator of signals for cell survival, proliferation, angiogenesis, and a number of metabolic insulin effects. Because of its growth-promoting effects, Akt plays a central role in tumorigenesis. A number of oncogenes and tumor suppressor genes act upstream of Akt to influence cancer progression.

The assay is designed for in vitro quantitative determination of human, mouse, and rat Akt/PKB protein in cell lysates.

Phospho-Akt/PKB (pThr ³⁰⁸) ELISA is designed to detect and quantify the levels of human, mouse and rat Akt/PKB protein phosphorylated on threonine 308. Akt/PKB is recruited from the cytosol to the plasma membrane and upon membrane localization, Akt undergoes a conformational change, which makes it accessible to phosphorylation at two kinase regulatory sites, threonine-308 of the kinase activation segment and serine-473 of the hydrophobic module. Carboxyl-terminal modulator protein (CTMP) binds to Akt/PKB a and reduces the activity by inhibiting phosphorylation at both threonine 308 and serine 473. These findings identify CTMP as a negative regulatory component of the pathway controlling PKB activity.

Akt/PKB ELISAs are a fast (total time 4 hours), sensitive (<0.1 ng/ml for non-phosphorylated and <0.8 units/mL for phosphorylated Akt/PKB) and specific (measure total or phosphorylated Akt/PKB with no crossreactivity with p38 MAPK, p42, JNK1 and others) alternative to immunoblotting or bioassays. The kit contains precoated plates and incubations are run at room temperature. No proprietary instrumentation is required.

The sensitivity is <0.8 units/mL, which is comparable to immunoblotting. The specificity is confirmed by peptide competition showing that only the phosphopeptide containing the phosphorylated threonine blocks the ELISA signal.