Amino-acid sequence analysis of proteins separated by one- and two-dimensional gel electrophoresis and electroblotted on polybase-coated glass-fiber sheets

Glass microfiber sheets to which guaternized ammonium polybases are adsorbed, were used as immobilizing membranes for protein electroblotting from sodium dodecylsulfate 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-boric acid buffer (50 mM each) provides the best conditions found so far. Until now, the NH2-terminal sequence of about one hundred different qlass-fiber immobilized proteins, polypeptides and glycoproteins, with molecular weight ranging from 10,000 to 150,000, have been successfully determined 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 qel..Moreover, we demonstrate that it is possible to determine the NH2~terminal amino acid sequence of the major proteins present in a total cellular extract, after separating them by 2D-PAGE followed by electroblotting on coated glass-fibeer. This allows one to analyse phenotypic alterations in cells or organisms at the elvel of the primary structure of the proteins. Based on the protein-sequence, a DNA-probe can be synthesized, which will allow the identification and isolation of the corresponding gene. This procedure was used to analyse the major proteins present in a total cellular extract of Nicotiana plumbaginifolia. Of the 10 proteins selected for analyses, eight yielded a sequence, ranging from 10 to 38 residues. Three of them are very homologous with other eukaryotic proteins: triosephosphate isomerase, Mn-superoxide dismutase and 1,3-beta-glucanase