A Radioisotope-free Oligosaccharyltransferase Assay Method

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2 Citations (Scopus)


Glycosylation of asparagine residues is widespread in Eukarya, and occurs in virtually all Archaea and some eubacterial species. A membrane-bound enzyme, oligosaccharyltransferase, catalyzes the transfer of an oligosaccharide chain from a sugar donor (lipid-linked oligosaccharide, LLO) to an asparagine residue in the consensus sequence, Asn-X-Ser/Thr (X ≠ Pro), in proteins. The in vitro oligosaccharyl transfer assay reaction mixture contains a detergent-solubilized oligosaccharyltransferase (OST), a sugar donor LLO, and a sugar acceptor peptide. Previous assay methods are problematic, in terms of the use of radioactive compounds and the cumbersome separation procedures using lectin binding or two-phase partitioning. Here, we describe a new oligosaccharyl transfer assay method, which is radioisotope-free and relies on a different separation mechanism. The glycopeptide products are separated from unreacted peptides by SDS-PAGE. A fluorescent dye is attached to the peptide substrate during custom peptide synthesis. The fluorescent imaging of the SDS-PAGE gels ensures high sensitivity and quantitative performance. The user-friendly PAGE format is particularly suitable for presentation in scientific papers. For illustrative applications, time-course and peptide library experiments are shown.

Original languageEnglish
Article numbere3186
Issue number5
Publication statusPublished - Mar 5 2019

All Science Journal Classification (ASJC) codes

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)
  • Plant Science


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