Babu, CVS Cho, SG Yoo, YS 2024-01-21T04:12:07Z 2024-01-21T04:12:07Z 2021-09-03 2005-10 0173-0835 https://pubs.kist.re.kr/handle/201004/136090 Signal transduction studies have indicated that Akt is essential for transducing the signals originating from extracellular stimuli. An exploration of the Akt signal transduction mechanism depends on the ability to assay its activation states by determining the ability of Akt to phosphorylate various substrates. This paper describes a CE-based kinase assay for Akt using a UV detection method. The RPRAATF peptide was used as the specific substrate to determine the Akt activity. Under the CE separation conditions used, the phosphorylated and nonphosphorylated forms of the RPRAATF peptide were rapidly resolved in the Akt reaction mixture within 20 min. Using this method for measuring the Akt activity, the incubation time for the Akt reactions as well as the kinetic parameters (Km) were examined. Furthermore, the developed method was applied to a PC12 cell system to assess the dynamics of the Akt activity by examining the effectiveness of the RPRAATF peptide substrate under various cytokine-stimulated environments. These results highlight the feasibility of the CE method, which is a simple and reliable technique for determining and characterizing various enzyme reactions particularly kinase enzymes. English WILEY PROTEIN-KINASE-B ASSAY Method development and measurements of endogenous serine/threonine Akt phosphorylation using capillary electrophoresis for systems biology Article 10.1002/elps.20050007 1 ELECTROPHORESIS, v.26, no.19, pp.3765 - 3772 ELECTROPHORESIS 26 19 3765 3772 scie scopus 000232579700020 2-s2.0-27244455420 Biochemical Research Methods Chemistry, Analytical Biochemistry & Molecular Biology Chemistry Article PROTEIN-KINASE-B ASSAY Akt capillary electrophoresis kinase assay PC12 cells protein kinease B signal transduction