UNIT 8.15 Release of Saccharides from Glycoconjugates
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Immunology
How to Cite
Powell, L. D., Varki, A. P. and Freeze, H. H. 2001. Release of Saccharides from Glycoconjugates. Current Protocols in Immunology. 9:IV:8.15:8.15.1–8.15.24.
- Published Online: 1 MAY 2001
- Published Print: MAR 1994
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Glycosidases are specific enzymes that can partially or completely remove sugar chains from cell-surface glycoconjugates. The enzymes can be exoglycosidases (which remove a terminal saccharide unit from an oligosaccharide chain), endoglycosidases (which cleave within an oligosaccharide chain, releasing an oligosaccharide fragment), or glycoamidases (which cleave between an oligosaccharide unit and its N-linkage to a protein). Commonly used examples of each enzyme are presented in this unit. Sialidase digestion of purified proteins is described and an Alternate Protocol details the application of the technique to intact cell suspensions. A support protocol describes testing sialidase activity in a variety of buffers. Basic protocols are detailed for the digestion of intact glycoproteins and glycopeptides by the most common endoglycosidases and glycoamidases: Endoglycosidase H (Endo H), Endoglycosidase F2 (Endo F2), and Peptide:N-glycosidase F (PNGase F). The applications described in the second and third support protocols employ these enzymes alone or as part of a sequential digestion. The second support protocol describes how to use partial digestions with one enzyme or sequential digestions with different enzymes to estimate the number or types of N-linked carbohydrate chains on a protein. The third support protocols describes sample preparation and digestion followed by gel-filtration chromatography to recover the released radiolabeled oligosaccharide chains for subsequent analysis.