Quantitation of glycoproteins on electroblots using the biotin-streptavidin complex.
A method for the detection and quantitation of glycoproteins on nitrocellulose electroblots is described. Protein mixtures may be solubilized in sodium dodecyl sulfate prior to labeling, which is especially useful when dealing with membrane proteins. Mild periodate oxidation produces aldehydes on the oligosaccharide moieties which are then specifically condensed with biotin aminocaproyl hydrazide. After polyacrylamide gel electrophoresis and transfer of proteins onto nitrocellulose membranes, biotinylated glycoproteins are detected with enzyme-linked streptavidin and quantitated by densitometric scanning. As little as 1 ng of alpha 1-acid glycoprotein can be detected by this method. The use of mild oxidation conditions renders the method highly selective for the detection of sialic acid-containing glycoproteins.
D001426:Bacterial Proteins; D001710:Biotin / Q000031:analogs & derivatives / Q000138:chemical synthesis; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D008565:Membrane Proteins / Q000097:blood; D009961:Orosomucoid / Q000032:analysis; D010084:Oxidation-Reduction; D019809:Streptavidin; D013696:Temperature
D001426:Bacterial Proteins; D006023:Glycoproteins; D008565:Membrane Proteins; D009961:Orosomucoid; C053312:biotin-6-aminocaproyl hydrazide; C053311:biotin hydrazide; D001710:Biotin; D003101:Collodion; D019809:Streptavidin