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.
Akt/PKB ELISA is designed to detect and quantify the levels of human, mouse and rat Akt/PKB protein, independent of its phosphorylation state. Akt is one of the downstream targets of PI-3 Kinase (PI3-K). Akt consist of three highly conserved isoforms, designated in humans as Akt1, Akt2, and Akt3. Each isoform consists of an N-terminal pleckstrin homology (PH) domain, which mediates lipid-protein or protein-protein interactions, and a C-terminal kinase catalytic domain. All isoforms respond in a similar fashion to various stimuli. Akt can be activated by growth factors and physiologic stimuli in a PI3-K-dependent manner. Activation of Akt kinase involves both membrane translocation and phosphorylation.
Features and Benefits
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 <0.1 ng/mL, which is ~2x more sensitive than immunoblotting. The assay is specific for total Akt/PKB and does not crossreact with p38 MAPK, p42 Erk1, p42 Erk2, JNK 1, human and rat insulin receptor, or human EGFR.