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

Kinoshita Eiji

Kinoshita-Kikuta Emiko

Takiyama Kei

Koike Tohru

Molecular and Cellular Proteomics

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).

Phosphoproteomics

Electroblotting analysis

Chemiluminescence detection

Phosphate-affinity electrophoresis

Phosphorylated protein

Phosphorylation

Biology [ 460 ]

eng

American Society for Microbiology

Copyright (c) 2006 by the American Society for Biochemistry and Molecular Biology.

[ISSN] 1535-9476

[DOI] 10.1074/mcp.T500024-MCP200

[NCID] AA11789524

[PMID] 16340016

[DOI] http://dx.doi.org/10.1074/mcp.T500024-MCP200 isVersionOf