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Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases
Published on: December 26, 2011
Identification and Characterization of GRASP55 O-GlcNAcylation
1College of Biomedicine and Health, Huazhong Agricultural University, Wuhan, China. xiaoyanz@mail.hzau.edu.cn.
Abstract:
O-GlcNAcylation is a posttranslational modification of proteins that adds a single sugar, β-N-acetylglucosamine (GlcNAc), to Ser/Thr residues. Extensive studies have been conducted to identify and characterize substrates of this modification but mostly focused on cytosolic and nuclear proteins. This chapter describes a detailed protocol used to determine the O-GlcNAcylation of GRASP55, the first O-GlcNAcylated Golgi protein identified, including how its O-GlcNAcylation level responds to glucose deprivation. In addition, this chapter also provides a detailed method to express and purify O-GlcNAcylated GRASP55 in bacteria for further in vitro functional assays. This protocol could be applied to other Golgi proteins that are potentially O-GlcNAcylated.
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