Inhibition of N-linked glycosylation

This unit describes the use of inhibitors in cultured cells to prevent N-linked glycosylation of proteins to yield glycoproteins containing missing or altered chains. This approach is useful for examining potential functional role(s) of oligosaccharides on specific proteins or intact cells. First, the optimal concentration of inhibitor for the experiment (i.e., highest nontoxic concentration) is determined by monitoring [(35)S]methionine incorporation as a measure of protein biosynthesis. The inhibitor's ability to inhibit oligosaccharide processing is then determined by analyzing cells labeled with [(3)H]mannose using TCA precipitation or endo H digestion. A support protocol details a method for concentrating proteins by acetone precipitation.