Detection of O -GlcNAc modifications on cardiac myofilament proteins

Abstract

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) b -Elimination and Michael Addition (BEMAD) for O -GlcNAc Site-Mapping. © Springer Science+Business Media New York 2013.