Identification of Glycoproteins on Nitrocellulose Membranes and Gels

Abstract

This chapter will focus on the identification of glycoproteins on gels and, in particular, after their immobilization by slot blotting onto nitrocellulose membranes. The procedures described have been optimized for the analysis of mucus glycoproteins (mucins) but should be applicable to other families of glycoprotein and to molecules that have been transferred to nitrocellulose by dot or Western blotting. Mucins are high-M _r glycoproteins that may contain up to 90% by weight carbohydrate primarily in the form of short chain O-linked oligosaccharides. A number of different mucins can be distinguished, some of which are located at the epithelial cell surface, whereas others are secreted and form mucus. The latter glycoproteins occur typically as macromolecular assemblies, some 5–30 × 10⁶ in mol wt and appear in electron microscopy as long filamentous threads often microns in length (1). Reduction of disulfide bonds yields a major fragment M _r 2–3 × 10⁶), which we term a subunit. Proteolytic digestion of subunits gives rise to large glycopeptides (M _r 300–500,000), which correspond to the very highly-substituted regions of the protein core.