Title [PROTOCOL] Determination of protein phosphorylation by polyacrylamide gel electrophoresis
Author Chang-Ro Lee1*, Young-Ha Park2, Huitae Min2, Yeon-Ran Kim2, and Yeong-Jae Seok2,3*
Address 1Department of Biological Sciences, Myongji University, Yongin 17058, Republic of Korea, 2School of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul 08826, Republic of Korea, 3Department of Biophysics and Chemical Biology, Seoul National University, Seoul 08826, Republic of Korea
Bibliography Journal of Microbiology, 57(2),93–100, 2019,
DOI 10.1007/s12275-019-9021-y
Key Words protein phosphorylation, electrophoretic mobility shift, SDS-PAGE, signal transduction, protein kinase
Abstract Phosphorylation is the most important modification for protein regulation; it controls many signal transduction pathways in all organisms. While several tools to detect phosphorylated proteins have been developed to study a variety of basic cellular processes involving protein phosphorylation, these methods have several limitations. Many proteins exhibit a phosphorylation-dependent electrophoretic mobility shift (PDEMS) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular mechanism responsible for this phenomenon has been elucidated recently. The method for detecting phosphorylated proteins can be simplified by the application of the PDEMS. Herein, we present a novel simple method to detect protein phosphorylation, which is based on the construction of a variant protein displaying a PDEMS. The PDEMS of proteins is caused by the distribution of negatively charged amino acids around the phosphorylation site, i.e. an electrophoretic mobility shift (EMS)-related motif (ΘX1-3ΘX1-3Θ, where Θ corresponds to an acidic or phosphorylated amino acid and X represents any amino acid). The EMS-related motif can be constructed by the introduction of a negative charge by phosphorylation; it results in the decreased binding of SDS to the proteins, consequently inducing the retardation of the mobility of the protein during SDS-PAGE. Based on these molecular analyses of the PDEMS, a protein with the EMSrelated motif is designed and used to determine the in vivo phosphorylation state of the protein. This method may be used as a general strategy to easily measure the ratio of protein phosphorylation in cells.