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
06/15/2013Pubmed ID
23765660DOI
10.1007/978-1-62703-465-4_12Scopus ID
2-s2.0-84880499724 (requires institutional sign-in at Scopus site) 5 CitationsAbstract
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 ASAuthor
Stephanie Olivier-Van Stichelen PhD Assistant Professor in the Biochemistry department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AcetylglucosamineAnimals
Antibodies
Blotting, Western
Electrophoresis, Polyacrylamide Gel
Enzyme Assays
Fluorescent Antibody Technique
Humans
Immunoprecipitation
N-Acetylglucosaminyltransferases
Silver Staining
Uridine Diphosphate