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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

Date

06/15/2013

Pubmed ID

23765660

DOI

10.1007/978-1-62703-465-4_12

Scopus ID

2-s2.0-84880499724   4 Citations

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.

Author List

Lefebvre T, Drougat L, Olivier-Van Stichelen S, Michalski JC, Vercoutter-Edouart AS

Author

Stephanie Olivier-Van Stichelen PhD Assistant Professor in the Biochemistry department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Acetylglucosamine
Animals
Antibodies
Blotting, Western
Electrophoresis, Polyacrylamide Gel
Enzyme Assays
Fluorescent Antibody Technique
Humans
Immunoprecipitation
N-Acetylglucosaminyltransferases
Silver Staining
Uridine Diphosphate