Unit

UNIT 8.14 Inhibition of N-Linked Glycosylation

  1. Leland D. Powell

Published Online: 1 MAY 2001

DOI: 10.1002/0471142735.im0814s09

Current Protocols in Immunology

Current Protocols in Immunology

How to Cite

Powell, L. D. 2001. Inhibition of N-Linked Glycosylation. Current Protocols in Immunology. 9:IV:8.14:8.14.1–8.14.9.

Author Information

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

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: MAR 1994

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 ability of the inhibitor to hinder oligosaccharide processing is then determined by analyzing cells labeled with [3H]mannose using TCA precipitation or endo H digestion. 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.