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Mobility shift detection of phosphorylation on large proteins using a Phos‐tag SDS‐PAGE gel strengthened with agarose
Author(s) -
Kinoshita Eiji,
KinoshitaKikuta Emiko,
Ujihara Hiromi,
Koike Tohru
Publication year - 2009
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200900020
Subject(s) - agarose , phos , polyacrylamide gel electrophoresis , molecular mass , microbiology and biotechnology , chemistry , gel electrophoresis , phosphorylation , kinase , biochemistry , phosphate , chromatography , biology , enzyme
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 μM 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).