Detection of O-GlcNAc modifications on cardiac myofilament proteins

In this chapter it is described a general method that has been used successfully by more than one laboratory interested in detecting O-GlcNAc in myofilament proteins. Alternative reagents for chemo-enzymatic or metabolic labeling will be indicated, as well as references for more details in alternative methods. The outline is divided into (1) Enrichment of O-GlcNAc Stoichiometry, (2) Cardiac Myofilament Protein Isolation, (3) SDS-PAGE, (4) "Reduction and Alkylation," (5) In-Gel Protein Digestion, (6) Chemo-enzymatic Labeling of O-GlcNAc Moieties (Click Chemistry), (7) Biotin Alkyne Tagging, (8) Strong Cation Exchange (SCX) and Streptavidin, and (9) β-Elimination and Michael Addition (BEMAD) for O-GlcNAc Site-Mapping.