Resolution of glycoproteins by a lectin gel-shift assay.
Gel-shift assays previously described in the literature are based on protein-protein or protein-DNA interactions. We show that carbohydrate-lectin interactions can be successfully used to alter the electrophoretic mobility of glycosylated, but not nonglycosylated, protein species in SDS-polyacrylamide gels. We were able to separate the two closely migrating mono- (95 kDa) and nonglycosylated (92 kDa) forms of a polytopic membrane protein, anion exchanger 1 (AE1), synthesized by cell-free translation or in transfected HEK293 cells. Concanavalin A was selected as the lectin due to the high mannose content of the oligosaccharide chain on AE1. Concanavalin A was either added 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 concanavalin A resulted in slower mobility of the monoglycosylated protein while the mobility of the nonglycosylated form was not altered. The shift in mobility was dependent on concentration of concanavalin A and the length of separating gel containing copolymerized concanavalin A. When a diglycosylated mutant of AE1 was tested, good separation was achieved at lower concentrations of concanavalin A. This lectin gel-shift assay allows the separation of N-glycosylated and nonglycosylated forms of the protein.
Popov
M
M
Medical Research Council Group in Membrane Biology, Department of Medicine, University ofToronto, Toronto, Ontario, M5S 1A8, Canada.
Li
J
J
Reithmeier
R A
RA
D000818:Animals; D017920:Antiporters / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D027963:Chloride-Bicarbonate Antiporters; D003208:Concanavalin A; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005069:Evaluation Studies as Topic; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D037102:Lectins; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate
D017920:Antiporters; D027963:Chloride-Bicarbonate Antiporters; D006023:Glycoproteins; D037102:Lectins; D011994:Recombinant Proteins; D003208:Concanavalin A; D012967:Sodium Dodecyl Sulfate