Nitrocellulose Filter Binding to Assess Binding of Glycosaminoglycans to Proteins

Abstract

This chapter outlines how the trapping of protein–saccharide complexes onto nitrocellulose filters can be used to explore the protein-binding potential of saccharide motifs. The method provides information on the size of saccharide epitopes recognized by proteins, as well as on affinity constants for these interactions. It may be applied in a preparative mode to yield saccharide fractions for structural analysis. The protocols that are presented describe the standard procedures for fragmentation and radiolabeling of heparin/heparan sulfate (HS). The isolation of HS-derived oligosaccharides to be tested as ligands is also focused. on Proteins are trapped onto nitrocellulose filters along with any bound saccharide ligand. Saccharides that do not bind to the protein are not retained by the filter. The filter-trapping procedure discussed is ideally suited for the screening of different proteins, available in limited quantities, regarding the ability to bind a defined radiolabeled saccharide ligand, a whole chain or an excised oligosaccharide fragment. Characterization of protein-binding saccharide domains that may be estimated by trapping experiments using series of oligosaccharides of different size is presented.