Unit

UNIT 17.10A Inhibition of N-Linked Glycosylation

  1. Leland D. Powell

Published Online: 1 MAY 2001

DOI: 10.1002/0471142727.mb1710as32

Current Protocols in Molecular Biology

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.

Author Information

  1. University of California San Diego, La Jolla, California

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: OCT 1995

Abstract

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.