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Updated: Jun 21, 2026

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag
Published on: December 14, 2017
Mobility shift detection of phosphorylation on large proteins using a Phos-tag SDS-PAGE gel strengthened with agarose
Eiji Kinoshita1, Emiko Kinoshita-Kikuta, Hiromi Ujihara
1Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8553, Japan. kinoeiji@hiroshima-u.ac.jp
Abstract:
We describe a novel technique of phosphate-affinity SDS-PAGE using Phos-tag to analyze large phosphoproteins with molecular masses of more than 200 kDa. The protein phosphoisotypes were clearly separated as up-shifted migration bands in a 3% w/v polyacrylamide gel containing 20 microM Phos-tag and 0.5% w/v agarose. In subsequent immunoblotting, the procedure permitted the determination of the phosphoisotypes of high-molecular-mass proteins, such as mTOR (289 kDa), ATM kinase (350 kDa), and 53BP1 (213 kDa).
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