Preparation of a neoglycoprotein using a homobifunctional reagent and its applicability for protein blotting and electron microscopy.

Abstract

A new method for the preparation of a neoglycoprotein (chemically mannosylated bovine serum albumin, D-Man.BSA) is described using the homobifunctional reagent divinylsulphone.D-Man.BSA purified by affinity chromatography on ConA-Sepharose 4B shows microheterogeneity as demonstrated by immunoaffinity electrophoresis with free ConA in the first-dimension gel. The dissociation constant K for the neoglycoprotein-ConA complex has been calculated to be 2.5.10(-5) M. Biotinylated D-Man.BSA is a useful reagent to detect carbohydrate binding proteins of L1210 leukemia cells on blots. The neoglycoprotein labelled with colloidal gold may be used to demonstrate L1210 cell surface D-Man binding proteins by preembedding electron microscopy.