WGA-based lectin affinity gel electrophoresis: A novel method for the detection of O-GlcNAc-modified proteins

• Published in: PloS one Vol. 12; no. 7; p. e0180714
• Main Authors: Kubota, Yuji, Fujioka, Ko, Takekawa, Mutsuhiro
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 07.07.2017; Public Library of Science (PLoS)
• Subjects: Acetylglucosamine - chemistry; Acetylglucosamine - metabolism; Affinity; Analysis; Biology and life sciences; Copolymerization; Electrophoresis; Electrophoretic mobility; Gel electrophoresis; Glycosylation; Kinases; Lectins; Lysates; Methods; Mobility; N-Acetylglucosamine; O-GlcNAcylation; Physical Sciences; Physiological aspects; Plasmids; Post-translation; Post-translational modifications; Properties; Protein Processing, Post-Translational; Proteins; Proteins - chemistry; Proteins - isolation & purification; Proteins - metabolism; Research and Analysis Methods; Residues; Science; Serine; Threonine; Translation; Wheat; Wheat germ; Wheat germ agglutinin; Wheat Germ Agglutinins - chemistry; Japan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0180714

Post-translational modification with O-linked β-N-acetylglucosamine (O-GlcNAc) occurs selectively on serine and/or threonine residues of cytoplasmic and nuclear proteins, and dynamically regulates their molecular functions. Since conventional strategies to evaluate the O-GlcNAcylation level of a specific protein require time-consuming steps, the development of a rapid and easy method for the detection and quantification of an O-GlcNAcylated protein has been a challenging issue. Here, we describe a novel method in which O-GlcNAcylated and non-O-GlcNAcylated forms of proteins are separated by lectin affinity gel electrophoresis using wheat germ agglutinin (WGA), which primarily binds to N-acetylglucosamine residues. Electrophoresis of cell lysates through a gel containing copolymerized WGA selectively induced retardation of the mobility of O-GlcNAcylated proteins, thereby allowing the simultaneous visualization of both the O-GlcNAcylated and the unmodified forms of proteins. This method is therefore useful for the quantitative detection of O-GlcNAcylated proteins.