In the ER, individual sugars are not added one-by-one to the protein to create an oligosaccharide side chain. Instead, a preformed, branched oligosaccharide containing a total of 14 sugars is attached en bloc to all proteins that carry the appropriate site for glycosylation.

The oligosaccharide is originally attached to a specialized lipid, called dolichol, in the ER membrane; it is then transferred to the amino (NH 2 ) group of an asparagine side chain on the protein, immediately after a target asparagine emerges in the ER lumen during protein translocation. Asparagines that are glycosylated are always present in the tripeptide sequences asparagine-X-serine or asparagine-X-threonine, where X can be almost any amino acid.

The addition takes place in a single enzymatic step that is catalyzed by a membrane-bound enzyme (an oligosaccharyl transferase) that has its active site exposed on the lumenal side of the ER membrane— which explains why cytosolic proteins are not glycosylated in this way.

A simple sequence of three amino acids, of which the target asparagine is one, defines which sites in a protein receive the oligosaccharide. Oligosaccharide side chains linked to an asparagine NH 2 group in a protein are said to be N-linked and this is by far the most common type of linkage found on glycoproteins.