Phosphate-binding Tag, a New Tool to Visualize Phosphorylated Proteins
摘要:
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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关键词:
Proteins Electrophoresis, Polyacrylamide Gel Magnetic Resonance Spectroscopy Blotting, Western Phosphorylation Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
DOI:
10.1074/mcp.T500024-MCP200
被引量:
年份:
2006




















































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