| High-Throughput Screening Method for Antigenic Proteins |
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Department of Bacteriology, Integrated Molecular Medicine, Nagoya University Graduate School of Medicine, Nagoya, JAPAN |
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| The identification of proteins on immunoblotting membranes has been one of the great challenges in molecular biology. We have refined the on-membrane digestion strategy to apply that method to immunoblotting. This strategy involved three key points: (1) Use polyvinylpyrrolidone (average molecular weight 40,000, PVP-40) as a blocking agent; (2) use a polyvinylidine difluoride (PVDF) membrane rather than a nylon membrane in the immunoblotting procedure; and (3) perform on-membrane digestion using an aqueous reaction system, eliminating the organic solvent. Liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry (LC-ESI-MS/MS) was used for protein identification, because several proteins were detected from a single band or spot on the immunoblotting membrane. The contaminated proteins were derived from the anti-serum or the background of individual organisms. This method should differentiate between target proteins and contaminants, based on the information on the organism, organ, or protein location. We expect this strategy to shed light on all molecular biological studies, especially in infectious and autoimmune disease research. | ||
| Key words: Immunoblotting, On-membrane digestion, LC-MS/MS, Antigen | ||
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