Premium
Detecting O‐GlcNAc using in vitro sulfation (607.1)
Author(s) -
Wu Zhengliang,
Robey mathew,
Tatge Timothy,
Leymarie Nancy,
Zou Yonglong,
Zaia Joseph
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.607.1
Subject(s) - sulfation , serine , glycosylation , threonine , biochemistry , chemistry , in vitro , phosphorylation
O‐GlcNAc glycosylation, the covalent attachment of N‐acetylglucosamine to serine and threonine residues of proteins, is a post‐translational modification that shares many features with protein phosphorylation. O‐GlcNAc is essential for cell survival and plays important roles in many biological processes (e.g., transcription, translation, cell division) and human diseases (e.g., diabetes, Alzheimer’s disease, cancer). However, detection of O‐GlcNAc is challenging. Here, a method for O‐GlcNAc detection using in vitro sulfation with two GlcNAc‐specific sulfotransferases, CHST2 and CHST4, and the radioisotope 35 S is described. Sulfation on free GlcNAc is first demonstrated, and then on O‐GlcNAc residues of peptides as well as nuclear and cytoplasmic proteins. It is also demonstrated that the sulfation on O‐GlcNAc is sensitive to OGT and OGA treatment. The labeled samples are separated on SDS‐PAGE and visualized by autoradiography. Overall, the method is sensitive, specific and convenient