Wheat Germ Agglutinin (WGA)-SDS-PAGE: A Novel Method for the Detection of O-GlcNAc-modified Proteins by Lectin Affinity Gel Electrophoresis

• Published in: Bio-protocol Vol. 8; no. 23; p. e3098
• Main Authors: Fujioka, Ko, Kubota, Yuji, Takekawa, Mutsuhiro
• Format: Journal Article
• Language: English
• Published: United States Bio-Protocol 05.12.2018; Bio-protocol LLC
• Subjects: Biology; Methods; Wheat germ agglutinin (WGA); Electrophoresis; O-GlcNAcylation; Post-translational modifications; Lectin affinity-gel
• ISSN: 2331-8325; 2331-8325
• DOI: 10.21769/bioprotoc.3098

Diverse cytoplasmic and nuclear proteins dynamically change their molecular functions by O-linked β-N-acetylglucosamine (O-GlcNAc) modification on serine and/or threonine residues. Evaluation of the O-GlcNAcylation level of a specific protein, however, needs multiple and time-consuming steps if using conventional methods ( , immune-purification, mass spectrometric analysis). To overcome this drawback, we developed the following easy and rapid method for detection of O-GlcNAcylated proteins of interest. An O-GlcNAc affinity gel layer containing wheat germ agglutinin (WGA), a GlcNAc-specific lectin, selectively induces retardation of the mobility of O-GlcNAcylated proteins during electrophoresis. This WGA-layer thereby separates O-GlcNAcylated and non-modified forms of proteins, allowing the detection and quantification of the O-GlcNAcylation level of these proteins. This new method therefore provides qualitative and quantitative analysis of O-GlcNAcylated proteins in a relatively shorter time compared to conventional methods.