UNIT 17.10A Inhibition of N-Linked Glycosylation
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Powell, L. D. 2001. Inhibition of N-Linked Glycosylation. Current Protocols in Molecular Biology. 32:II:17.10A:17.10.1–17.10.9.
- Published Online: 1 MAY 2001
- Published Print: OCT 1995
Treatment of cells with inhibitors of the enzymes that synthesize N-linked oligosaccharide chains results in production of glycoproteins containing missing or altered chains. This approach is useful for examining potential functional role(s) of this class of oligosaccharides on specific proteins or intact cells. This unit describes the use of inhibitors to prevent N-linked glycosylation of proteins in cultured cells. First, the optimal concentration of inhibitor for the experiment (i.e., highest nontoxic concentration) is determined by monitoring [35S]methionine incorporation as a measure of protein biosynthesis. The inhibitor's ability to inhibit oligosaccharide processing is then determined by analyzing cells labeled with [HH]mannose using TCA precipitation or endo H digestion (unit 13). Further suggestions are given on how to use methods for identifying a specific glycoprotein (if available) to measure the effect of the inhibitor on its N-linked oligosaccharide chains. A support protocol details a method for concentrating proteins by acetone precipitation.