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Originally published In Press as doi:10.1074/mcp.T500024-MCP200 on December 11, 2005.
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Molecular & Cellular Proteomics 5:749-757, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Technology

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

Eiji Kinoshita{ddagger},§,¶, Emiko Kinoshita-Kikuta{ddagger}, Kei Takiyama{ddagger} and Tohru Koike{ddagger},§,||

From the {ddagger} 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).

To whom correspondence may be addressed. Tel.: 81-82-257-5281; Fax: 81-82-257-5336; E-mail: kinoeiji{at}hiroshima-u.ac.jp


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