WGA-SDS-PAGE: A lectin-based, new affinity gel electrophoresis for quantitative analysis of O-GlcNAc proteins

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  • Kubota Yuji
    Division of Cell Signaling and Molecular Medicine, Institute of Medical Science, The University of Tokyo
  • Fujioka Ko
    Division of Cell Signaling and Molecular Medicine, Institute of Medical Science, The University of Tokyo
  • Takekawa Mutsuhiro
    Division of Cell Signaling and Molecular Medicine, Institute of Medical Science, The University of Tokyo

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  • O-GlcNAc化蛋白質の検出と定量的解析を可能にするレクチン親和性ゲル電気泳動法の開発と応用

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Abstract

<p>O-linked β-N-acetylglucosamine (O-GlcNAc) modification is one of protein post-translational modifications in multicellular eukaryotes. This modification occurs selectively on serine and/or threonine residues of specific cytoplasmic and nuclear proteins and dynamically modulates their molecular functions. However, conventional methods for the evaluation of the physiological O-GlcNAcylation level (e.g., immune-purification, mass spectrometric analysis) of a specific protein need time-consuming and complicated steps. We therefore developed a rapid and easy method for the detection and quantification of an O-GlcNAcylated protein. Here, we describe the principal and experimental procedure of the novel affinity gel electrophoresis that separates O-GlcNAcylated and non-O-GlcNAcylated forms of proteins. The polyacrylamide-conjugated wheat germ agglutinin (WGA), which preferentially binds to N-acetylglucosamine residues, selectively induces retardation of the mobility of O-GlcNAcylated proteins during electrophoresis and thereby allows the visualization of both O-GlcNAc-modified and unmodified forms of specific proteins. Thus, our method, termed WGA-SDS-PAGE, provides a useful tool for quantitative monitoring of protein O-GlcNAcylation.</p>

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