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Submitted on September 20, 2005
Revised on November 4, 2005
Accepted on December 11, 2005

Phosphate-binding tag: A new tool to visualize phosphorylated proteins

Eiji Kinoshita, Emiko Kinoshita-Kikuta, Kei Takiyama, and Tohru Koike

Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Hiroshima 734-8551

Corresponding Author: kinoeiji{at}hiroshima-u.ac.jp

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 enhanced chemiluminescence (ECL) system using biotin-pendant Zn2+–Phos-tag and horseradish peroxidase-conjugated streptavidin (HRP–SA). 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 the phosphate-affinity electrophoresis (Mn2+–Phos-tag SDS-PAGE).


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