Antibodies and activity measurements for the detection of O-GlcNAc transferase and assay of its substrate, UDP-GlcNAc

  • Methods Mol Biol. 2013;1022:147-59. doi: 10.1007/978-1-62703-465-4_12.
Tony Lefebvre  1 ,  Ludivine Drougat ,  Stephanie Olivier-Van Stichelen ,  Jean-Claude Michalski ,  Anne-Sophie Vercoutter-Edouart
Affiliations
  • 1. Unit of Structural and Functional Glycobiology, University of Lille 1, Villeneuve d'Ascq, France.
PMID: 23765660 DOI: 10.1007/978-1-62703-465-4_12
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.

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