Resolution of glycoproteins by a lectin gel-shift assay

Gel-shift assays previously described in the literature are based on protei n-protein or protein-DNA interactions. We show that carbohydrate-lectin int eractions can be successfully used to alter the electrophoretic mobility of glycosylated, but not nonglycosylated, protein species in SDS-polyacrylami de gels. We were able to separate the two closely migrating mono- (95 kDa) and nonglycosylated (92 kDa) forms of a polytopic membrane protein, anion e xchanger 1 (AE1), synthesized by cell-free translation or in transfected HE K293 cells. Concanavalin A was selected as the lectin due to the high manno se content of the oligosaccharide chain on AE1. Concanavalin A was either a dded to the samples prior to loading or copolymerized in a top layer of the separating gel, the latter being the method of choice. The presence of con canavalin A resulted in slower mobility of the monoglycosylated protein whi le the mobility of the nonglycosylated form was not altered. The shift in m obility was dependent on concentration of concanavalin A and the length of separating gel containing copolymerized concanavalin A. When a diglycosylat ed mutant of AE1 was tested, good separation was achieved at lower concentr ations of concanavalin A. This lectin gel-shift assay allows the separation of N-glycosylated and nonglycosylated forms of the protein. (C) 2000 Acade mic Press.