Antibodies and Activity Measurements for the Detection of O-GlcNAc Transferase and Assay of its Substrate, UDP-GlcNAc
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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.
