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Molecular & Cellular Proteomics 5:749-757, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Technology

Phosphate-binding Tag, a New Tool to Visualize Phosphorylated Proteins^*,S

Eiji Kinoshita^,,¶, Emiko Kinoshita-Kikuta, Kei Takiyama and Tohru Koike^,,||

From the Department of Functional Molecular Science, Graduate School of Biomedical Sciences, and Frontier Center for Microbiology, Hiroshima University, Kasumi 1-2-3, Hiroshima 734-8551, Japan

We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn²⁺ or Mn²⁺) complexes as novel phosphate-binding tag (Phos-tag) molecules. Both Zn²⁺- and Mn²⁺-Phos-tag molecules preferentially capture phosphomonoester dianions bound to Ser, Thr, and Tyr residues. One method is based on an ECL system using biotin-pendant Zn²⁺-Phos-tag and horseradish peroxidase-conjugated streptavidin. We demonstrate the electroblotting analyses of protein phosphorylation status by the phosphate-selective ECL signals. Another method is based on the mobility shift of phosphorylated proteins in SDS-PAGE with polyacrylamide-bound Mn²⁺-Phos-tag. Phosphorylated proteins in the gel are visualized as slower migration bands compared with corresponding dephosphorylated proteins. We demonstrate the kinase and phosphatase assays by phosphate affinity electrophoresis (Mn²⁺-Phos-tag SDS-PAGE).

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