Journal of Microbiology

, Volume 57, Issue 2, pp 93–100 | Cite as

Determination of protein phosphorylation by polyacrylamide gel electrophoresis

  • Chang-Ro LeeEmail author
  • Young-Ha Park
  • Huitae Min
  • Yeon-Ran Kim
  • Yeong-Jae SeokEmail author


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.


protein phosphorylation electrophoretic mobility shift SDS-PAGE signal transduction protein kinase 


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Copyright information

© The Microbiological Society of Korea and Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Biological SciencesMyongji UniversityYonginRepublic of Korea
  2. 2.School of Biological Sciences and Institute of MicrobiologySeoul National UniversitySeoulRepublic of Korea
  3. 3.Department of Biophysics and Chemical BiologySeoul National UniversitySeoulRepublic of Korea

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