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  • Detailed structural analysis of N-glycans released from glycoproteins in SDS-PAGE gel bands using HPLC combined with exoglycosidase array digestions.

Detailed structural analysis of N-glycans released from glycoproteins in SDS-PAGE gel bands using HPLC combined with exoglycosidase array digestions.

Methods in molecular biology (Clifton, N.J.) (2006-10-31)
Louise Royle, Catherine M Radcliffe, Raymond A Dwek, Pauline M Rudd
RESUMO

In contrast to the linear sequences of protein and DNA, oligosaccharides are branched structures. In addition, almost all glycoproteins consist of a heterogeneous collection of differently glycosylated variants. Glycan analysis therefore requires high-resolution separation techniques that can provide detailed structural analysis, including both monosaccharide sequence and linkage information. This chapter describes how a combination of high-performance liquid chromatography (HPLC) and exoglycosidase enzyme array digestions can deliver quantitative glycan analysis of sugars released from glycoproteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel bands by matching HPLC elution positions with a database of standard glycans.

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Acetonitrile, for HPLC, for UV, ≥99.9% (GC)