Enzyme-based detection of glycoproteins on blot transfers using avidin-biotin technology |
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| Authors: | E A Bayer H Ben-Hur M Wilchek |
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| Institution: | 1. College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu Province 210023, People’s Republic of China;2. Laboratory of Translational Medicine, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing, Jiangsu Province 210028, People’s Republic of China;3. Department of Nuclear Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province 210029, People’s Republic of China;4. Bijie Institute of Traditional Chinese Medicine, Bijie, Guizhou Province 551700, People’s Republic of China;5. Department of Natural Medicinal Chemistry and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, Jiangsu Province 210009, People’s Republic of China;6. Shandong Academy of Medical Sciences, Jinan, Shandong 250062, People’s Republic of China;7. Theragnostic LaboratoryCampus Gasthuisberg, KU LeuvenLeuven3000Belgium |
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| Abstract: | 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. |
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