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Chapitre D'ouvrage Année : 2013

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

Résumé

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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Dates et versions

hal-03173338 , version 1 (18-03-2021)

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Tony Lefebvre, Ludivine Drougat, Stéphanie Olivier-van Stichelen, Jean-Claude Michalski, Anne-Sophie Vercoutter-Edouart. Antibodies and activity measurements for the detection of O-GlcNAc transferase and assay of its substrate, UDP-GlcNAc. Brockhausen, Inka. Glycosyltransferases : Methods and Protocols, 1022, Springer, pp.147-159, 2013, 978-1-62703-465-4. ⟨10.1007/978-1-62703-465-4_12⟩. ⟨hal-03173338⟩

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