Abstract
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.
Highlights
Protein post-translational modifications (PTMs) regulate various properties of proteins such as stability, subcellular localization, and catalytic activity
As O-GlcNAcylated proteins pass through the wheat germ agglutinin (WGA)-containing layer of the gel, the O-GlcNAc residues interact with the immobilized-WGA, resulting in retardation of their mobility
To evaluate the efficacy of WGA-SDS-PAGE for the separation of O-GlcNAcylated proteins, we used Tab1, which is an activator of TAK1/MAP3K7 [17], as a representative O-GlcNAcylatable protein [18]
Summary
Protein post-translational modifications (PTMs) regulate various properties of proteins such as stability, subcellular localization, and catalytic activity. Immunopurified HA-Tab1 was first separated in the absence of the WGA-gel layer by using standard SDS-PAGE, and the O-GlcNAcylation level of Tab1 was assessed by immunoblotting using either an anti-O-GlcNAc antibody (RL2) or WGA-HRP.
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