Differential Detection of O-GlcNAcylated proteins in the heart using antibodies

Thousands of mammalian intracellular proteins are dynamically modified by O-linked β−N-acetylglucosamine (O-GlcNAc). Global changes in O-GlcNAcylation have been associated with the development of cardiomyopathy, heart failure, hypertension, and neurodegenerative disease. Levels of O-GlcNAc in cells and tissues can be detected using numerous approaches; however, immunoblotting using GlcNAc-specific antibodies and lectins is commonplace. The goal of this study was to optimize the detection of O-GlcNAc in heart lysates by immunoblotting. Using a combination of tissue fractionation, immunoblotting, and galactosyltransferase labeling, as well as hearts from wild-type and O-GlcNAc transferase transgenic mice, we demonstrate that contractile proteins in the heart are differentially detected by two commercially available antibodies (CTD110.6 and RL2). As CTD110.6 displays poor reactivity toward contractile proteins, and as these proteins represent a major fraction of the heart proteome, a better assessment of cardiac O-GlcNAcylation is obtained in total tissue lysates with RL2. The data presented highlight tissue lysis approaches that should aid the assessment of the cardiac O-GlcNAcylation by immunoblotting. [Display omitted] •The O-GlcNAc antibody CTD110.6 does not react well with heart contractile proteins.•The O-GlcNAc antibody RL2 does react well with heart contractile proteins.•RL2 is preferred to CTD110.6 when analyzing O-GlcNAc in cardiac lysates.•Analysis of fractionated hearts improves the detection of O-GlcNAc.