Biotinylated Carbohydrate Markers -A Novel Tool for Lectin Research

Abstract

One of the key obstacles in lectin research is the lack of specific techniques for their detection. Novel markers, biotin-labeled carbohydrates, could contribute to overcome this problem. Being at least 10 times more sensitive than neoglycoproteins in the membranescreening assays, they also enable direct detection of lectins in complex mixtures. The markers were synthesized by linking biotin to one, and a carbohydrate (galactose or glucose) to the other amino group of (the amino acid) lysine. After synthesis the markers were chromatographically purified on lectin (RCA for galactose marker, ConA for glucose marker) and avidin affinity columns. The applicability of the markers to detect lectins was demonstrated e.g. with sponge extracts (from Geodia cydonium). Following incubation with biotin-labeled carbohydrates covalent cross-linking between lectins and markers was induced by UV radiation. After transfer to the blotting membrane, lectins were detected with deglycosylated antibiotin antibody labeled with alkaline phosphatase. Besides for the cross-linking technique, the biotinylated carbohydrate markers were also used for detection of lectins on the nitrocellulose membrane in gene library screening and slot blotting.