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

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)