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. |