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Related Experiment Videos

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

Eiji Kinoshita1, Emiko Kinoshita-Kikuta, Kei Takiyama

  • 1Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima 734-8551, Japan.

Molecular & Cellular Proteomics : MCP
|December 13, 2005
PubMed
Summary

We developed novel phosphate-binding tag (Phos-tag) molecules for visualizing phosphorylated proteins. These Phos-tags enable sensitive detection using electrochemiluminescence or mobility shift assays in SDS-PAGE.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

Background:

  • Protein phosphorylation is a critical post-translational modification regulating cellular processes.
  • Accurate visualization of phosphorylated proteins is essential for studying kinase and phosphatase activities.
  • Existing methods for detecting protein phosphorylation can be limited in sensitivity or scope.

Purpose of the Study:

  • To introduce novel phosphate-binding tag (Phos-tag) molecules for visualizing phosphorylated proteins.
  • To develop two distinct methods for protein phosphorylation detection using these Phos-tags.
  • To demonstrate the utility of Phos-tags in analyzing protein phosphorylation status and enzyme activities.

Main Methods:

  • Synthesis and application of alkoxide-bridged dinuclear metal complexes (Zn(2+) and Mn(2+)) as Phos-tags.

Related Experiment Videos

  • Development of an electrochemiluminescence (ECL) system using biotin-pendant Zn(2+)-Phos-tag and streptavidin-horseradish peroxidase.
  • Implementation of a mobility shift assay in SDS-PAGE using polyacrylamide-bound Mn(2+)-Phos-tag for phosphate affinity electrophoresis.

    • Main Results:

    • Zn(2+)- and Mn(2+)-Phos-tag molecules selectively bind phosphomonoester dianions on Ser, Thr, and Tyr residues.
    • The ECL method enabled sensitive detection of protein phosphorylation status via electroblotting.
    • Mn(2+)-Phos-tag SDS-PAGE clearly distinguished phosphorylated from dephosphorylated proteins by altered migration.
    • Phos-tag based assays successfully demonstrated kinase and phosphatase activities.

    Conclusions:

    • Phos-tag technology provides novel and effective tools for visualizing protein phosphorylation.
    • The developed methods offer high sensitivity and specificity for detecting phosphorylation events.
    • Phos-tag applications extend to biochemical assays for kinase and phosphatase activity determination.