Enzyme-based detection of glycoproteins on blot transfers using avidin-biotin technology.
Avidin-biotin technology has been employed for the improved nonradioactive detection of glycoproteins on blots. Periodate oxidation of samples on blots converts the glycoprotein-based carbohydrate residues to the corresponding aldehydes. The latter undergo interaction with preformed complexes consisting of either avidin hydrazide or streptavidin hydrazide combined with biotinylated alkaline phosphatase. The sensitivity of the new assay exceeds the previously described enzyme hydrazide method by a factor of at least 10. The approach can be rendered selective for sialoglycoproteins, and approximately 12 sugar-containing bands could be observed in erythrocyte membrane preparations. Problems of nonspecific binding and high levels of background label were alleviated using a nonglycosylated basic protein (lysozyme) for quenching.
D000469:Alkaline Phosphatase; D001360:Avidin; D001426:Bacterial Proteins; D001710:Biotin; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D010084:Oxidation-Reduction; D010504:Periodic Acid; D012795:Sialoglycoproteins / Q000032:analysis; D019809:Streptavidin
D001426:Bacterial Proteins; D006023:Glycoproteins; D012795:Sialoglycoproteins; D010504:Periodic Acid; D001360:Avidin; D001710:Biotin; D003101:Collodion; D019809:Streptavidin; C009288:metaperiodate; D000469:Alkaline Phosphatase