A Support Protocol details a method for concentrating proteins by acetone precipitation.
N LINKED GLYCOSYLATION HOW TO
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. The ability of the inhibitor to hinder oligosaccharide processing is then determined by analyzing cells labeled with mannose using TCA precipitation or endo H digestion. First, the optimal concentration of inhibitor for the experiment (i.e., highest nontoxic concentration) is determined by monitoring methionine incorporation as a measure of protein biosynthesis. Almost all membrane proteins undergo folding and maturation in the ER, where N-linked glycosylation usually fulfils a critical role in biosynthetic quality control 13, 14. N-linked glycosylation is the most frequent protein modification in eukaryotes. This unit describes the use of inhibitors to prevent N-linked glycosylation of proteins in cultured cells. Waker and collaborators identified the native glycosylation capability in Campylobacter jejuni and demonstrated that their N-linked glycosylation pathway. Its structure is the same in animals, plants and single. This approach is useful for examining potential functional role(s) of this class of oligosaccharides on specific proteins or intact cells. N-linked glycosylation sites (N-X-S or N-X-T) are indicated by stick figures on the protein (dark line). Biosynthesis of N-linked glycoproteins First a lipid-linked oligosaccharide precursor is synthesized. Treatment of cells with inhibitors of the enzymes that synthesize N-linked oligosaccharide chains results in production of glycoproteins containing missing or altered chains.
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