Protein-Blotting from SDS-Polyacrylamide Gels on Glass-Fiber Sheets Coated with Quaternized Ammonium Polybases

Summary

Glass microfiber sheets to which quaternized ammonium polybases are adsorbed, were used as immobilizing membranes for protein electroblotting from sodium dodecylsulfate-containing polyacrylamide gels. We have studied blotting efficiencies for various proteins, using different types of polybases and transfer buffers. We found that coating the glass-fiber sheets with poly (4-vinyl-N-methylpyridinium iodide) and carrying out the electrotransfer in Tris (hydroxymethyl) aminomethane-borate buffer, 50 mM, pH 8.5, provides the best conditions found so far. Here, Whatmann GF/F glass-fiber sheets exhibit binding capacities for serum albumin and actin which are higher than 50 micrograms/cm². So far, about fifty different glass-fiber-immobilized proteins, polypeptides and glycoproteins, with Mr ranging from 10,000 to 125,000, have been successfully sequenced at their amino-terminus using classical gas-phase sequence analysis. The technique was found to be especially suitable for partial sequence analysis of proteins which were first enriched before final purification on gels. As an illustration, we show a sequence analysis covering more than 28 residues of human serum albumin which was purified from serum by two-dimensional gel electrophoresis and electroblotted on high capacity sheets.