Direct carbohydrate analysis of glycoproteins electroblotted onto polyvinylidene difluoride membrane from sodium dodecyl sulfate-polyacrylamide gel.
A procedure for the carbohydrate analysis of glycoproteins electrotransferred to a polyvinylidene difluoride membrane is described. The glycoproteins (plant lectins, transferrin, and vitronectin) were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto a membrane. Each of the glycoprotein bands visualized by staining with Coomassie brilliant blue R-250 was excised from the membrane and subjected to direct hydrolysis either in 2.5 M trifluoroacetic acid at 100 degrees C for 6 h for neutral sugars and hexosamines, or in 0.05 M H2SO4 at 80 degrees C for 1 h for sialic acids. The hydrolysate obtained was analyzed for neutral sugars, hexosamines, and sialic acids independently by three different systems of high-performance liquid chromatography. The analytical values were reproducible with reasonable accuracy and agreed with those expected with recoveries of 57-66%. The method was successfully applied to a mannose-specific lectin of Sophora japonica bark, which is composed of four different subunits that aggregate sugar specifically. Because the four subunits could be separated by SDS-PAGE alone, the method proved useful for determining their carbohydrate compositions. Three of them were shown to contain carbohydrates typical of N-linked oligosaccharides of plant origin, which agreed well with the results of the binding assay carried out on a membrane using various horseradish peroxidase-labeled lectins.
Ogawa
H
H
Department of Chemistry, Faculty of Science, Ochanomizu University, Tokyo, Japan.
Ueno
M
M
Uchibori
H
H
Matsumoto
I
I
Seno
N
N
D002241:Carbohydrates / Q000737:chemistry*; D003208:Concanavalin A / Q000031:analogs & derivatives; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry*; D006595:Hexosamines / Q000737:chemistry; D006735:Horseradish Peroxidase; D037102:Lectins / Q000737:chemistry; D037241:Mannose-Binding Lectins; D008567:Membranes, Artificial; D019158:N-Acetylneuraminic Acid; D037121:Plant Lectins; D010944:Plants / Q000032:analysis*; D011145:Polyvinyls; D012794:Sialic Acids / Q000737:chemistry; D013194:Staining and Labeling; D005465:Fluorocarbon Polymers
D002241:Carbohydrates; D003208:Concanavalin A; D006023:Glycoproteins; D006595:Hexosamines; D006735:Horseradish Peroxidase; D037102:Lectins; D037241:Mannose-Binding Lectins; D008567:Membranes, Artificial; D019158:N-Acetylneuraminic Acid; D037121:Plant Lectins; D011145:Polyvinyls; D012794:Sialic Acids; C045779:concanavalin A-horseradish peroxidase conjugate; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers