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Optimized phos-tag mobility shift assay for the detection of protein phosphorylation in planta
J Plant Biotechnol 2018;45:322-327
Published online December 31, 2018
© 2018 The Korean Society for Plant Biotechnology.

Shah Hussain, Nhan Thi Nguyen, Xuan Canh Nguyen, Chae Oh Lim, Woo Sik Chung

Division of Applied Life Science (BK21 Plus program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, 52828, Republic of Korea
Faculty of Biotechnology, Vietnam National University of Agriculture, Hanoi, Vietnam
Division of Life Science, Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, 52828, Republic of Korea
Correspondence to: email: chungws@gnu.ac.kr
Received November 15, 2018; Revised November 21, 2018; Accepted November 21, 2018.
cc This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Post-translational modification of proteins regulates signaling cascades in eukaryotic system, including plants. Among these modifications, phosphorylation plays an important role in modulating the functional properties of proteins. Plants perceive environmental cues that directly affect the phosphorylation status of many target proteins. To determine the effect of environmentally induced phosphorylation in plants, in vivo methods must be developed. Various in vitro methods are available but, unlike in animals, there is no optimized methodology for detecting protein phosphorylation in planta. Therefore, in this study, a robust, and easy to handle Phos-Tag Mobility Shift Assay (PTMSA) is developed for the in vivo detection of protein phosphorylation in plants by empirical optimization of methods previously developed for animals. Initially, the detection of the phosphorylation status of target proteins using protocols directly adapted from animals failed. Therefore, we optimized the steps in the protocol, from protein migration to the transfer of proteins to PVDF membrane. Supplementing the electrophoresis running buffer with 5 mM NaHSO3 solved most of the problems in protein migration and transfer. The optimization of a fast and robust protocol that efficiently detects the phosphorylation status of plant proteins was successful. This protocol will be a valuable tool for plant scientists interested in the study of protein phosphorylation.
Keywords : Electrophoresis mobility shift assay, Phos-tag, Plant, Protein kinases, Protein phosphorylation


December 2018, 45 (4)
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