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      Phosphate-binding tag, a new tool to visualize phosphorylated proteins.

      Molecular & Cellular Proteomics : MCP

      Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Electrophoresis, Polyacrylamide Gel, Magnetic Resonance Spectroscopy, Phosphorylation, Proteins, isolation & purification, metabolism, Blotting, Western

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          Abstract

          We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn(2+) or Mn(2+)) complexes as novel phosphate-binding tag (Phos-tag) molecules. Both Zn(2+)- and Mn(2+)-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 Zn(2+)-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 Mn(2+)-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 (Mn(2+)-Phos-tag SDS-PAGE).

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          Journal
          16340016
          10.1074/mcp.T500024-MCP200

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