Detection of O-GlcNAc modifications on cardiac myofilament proteins.
Genaro A Ramirez-Correa, Isabel Martinez Ferrando, Gerald Hart, Anne Murphy
Division of Cardiology, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Methods in molecular biology (Clifton, N.J.) 2013In 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.
Citation
Genaro A Ramirez-Correa, Isabel Martinez Ferrando, Gerald Hart, Anne Murphy. Detection of O-GlcNAc modifications on cardiac myofilament proteins. Methods in molecular biology (Clifton, N.J.). 2013;1005:157-68
PMID: 23606256
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