Microplate assays to measure total and phospho-AKT in whole cells.
Thermo Scientific Pierce AKT In-Cell ELISA Kits are colorimetric and fluorescent microplate assays optimized to measure total and phosphorylated Akt family of serine/threonine-specific protein kinases (phospho-AKT) in whole cells.
These ELISA kits for whole cells include a mouse monoclonal anti-AKT antibody, rabbit monoclonal anti-phospho-(Ser473) AKT antibody, detection reagents and all buffers required for either colorimetric or near infrared detection. The AKT In-Cell ELISA Kits enable simultaneous detection of both total and phospho-(Ser473) AKT.
Measures total AKT and phospho-AKT (Ser473) in whole cells using 96-well or 384-well microplates
colorimetric detection kit for use with standard microplate readers
near infrared fluorescent detection kit for use with IR imaging system
Whole-cell detection requires no cell lysis, protein extraction or special sample preparation
Provides better data and higher throughput than quantitative Western blotting
Less costly (one well vs. one lane) than Western blotting
Stimulation with EGF causes increased AKT phosphorylation A431 cells. Relative total and phospho-AKT levels were measured in A431 cells using the AKT (A) Colorimetric and (B) Near IR In-Cell ELISA Kits. Cells were serum-starved overnight and stimulated with 100ng/ml EFG for five minutes immediately prior to fixation. Total AKT was not significantly different between experiments or treatments. Note: Absolute absorbance values cannot be directly compared between phospho and total AKT experiments because they use antibodies with different sensitivities.
Overview of AKT and phosphorylation of AKT:
AKT proteins, also know as protein kinases B (PKB), are important for cell signaling in mammals. AKT1, 2, and 3 are serine/threonine-specific kinases. AKT1 is involved in cell survival and inhibition of apoptosis and is implicated in human cancers as well as growth regulation in mice. AKT2 has role in the insulin signaling pathway and has been shown to be linked to a diabetic phenotype in mice. AKT3 is highly expressed in the brain and knock-out of AKT3 results in reduced brain mass in mice. AKT proteins have a PH domain which binds to phosphoinositides which are phosphorylated by PI3-kinases. When located at the cell membrane and bound to PIP3, AKT protein can be phosphorylated by mTORC2 (Ser 473) allowing subsequent phosphorylation by PDPK1 (Thr 308). This activates AKT kinase activity which leads to the regulation of downstream targets. The AKT In-Cell ELISA Kits enable simultaneous detection of both total and phospho-(Ser473) AKT.