title: Antibodies and activity measurements for the detection of O-GlcNAc transferase and assay of its substrate, UDP-GlcNAc.
abstract: Since the discovery of O-GlcNAc modification (O-GlcNAcylation) 20 years ago, much attention has been given to OGT (O-GlcNAc transferase), the unique enzyme responsible for the nuclear and cytosolic O-GlcNAcylation processes. This review focuses on protocols that are routinely used to analyze OGT expression and activity. First are detailed techniques using rabbit polyclonal anti-OGT antibodies, namely, Western blot, (co-)immunoprecipitation, and immunofluorescence. We also describe the measurement of OGT activity by using synthetic peptides as acceptors and radiolabeled UDP-GlcNAc. Finally, a sensitive HPAEC-based technique to measure the cellular content of UDP-GlcNAc, the donor substrate of OGT, is described in detail.
authors.affiliation: Unit of Structural and Functional Glycobiology, University of Lille 1, Villeneuve d'Ascq, France.
authors.forename: Tony
authors.initials: T
authors.lastname: Lefebvre
authors.forename: Ludivine
authors.initials: L
authors.lastname: Drougat
authors.forename: Stephanie
authors.initials: S
authors.lastname: Olivier-Van Stichelen
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Michalski
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Vercoutter-Edouart
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000032:analysis; D015153:Blotting, Western / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D057075:Enzyme Assays / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000379:methods; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D017351:N-Acetylglucosaminyltransferases / Q000032:analysis* / Q000378:metabolism*; D016622:Silver Staining / Q000379:methods; D014530:Uridine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D000906:Antibodies; D014530:Uridine Diphosphate; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; D000117:Acetylglucosamine