TRIS TRICINE AND TRIS BORATE BUFFER SYSTEMS PROVIDE BETTER ESTIMATES OF HUMAN MESOTHELIAL CELL INTERMEDIATE FILAMENT PROTEIN MOLECULAR-WEIGHTS THAN THE STANDARD TRIS GLYCINE SYSTEM

Human mesothelial cells contain a number of well defined intermediate filament proteins (IFs) that have been completely sequenced including vimentin and the cytokeratins (K7, K8, K18, and K19). The electrophoretic migration of these IFs was monitored as a function of second dimension gel buffer composition using various systems including Tris-glycine (pH 8.3 or 9.2), Tris-glycine with 20% methanol, Tris-borate, Tris-tricine, and sodium phosphate. All of the second dimension buffer chemistries yielded patterns of sufficient resolution to identify the major cytoskeletal proteins but differed in the relative mobilities of the IFs. Using gene sequence calculated molecular weight data, the major cytoskeletal polypeptides of human mesothelial cells were ranked from highest molecular weight to lowest molecular weight. This rank order of sequence calculated molecular weights was then compared to the rank order determined from the actual migration of the polypeptides in the different gel systems. With the Tris-tricine and the Tris-borate gel systems as well as gene sequence data, K8 = vimentin > β-tubulin = K7 > K18 > K19 > actin. With the pH 8.3 and 9.2 Tris-glycine systems, as well as the sodium phosphate gel system, the rank order of the polypeptides did not correspond to gene sequence data. Adding 20% methanol to the Tris-glycine system resulted in IF migration that more closely corresponded to the gene sequence derived data. Migration position of the IFs depended upon the temperature of the second dimension separation as well. In mesothelial cells, the migration of a total of 15-25% of the polypeptides was influenced by differing buffer systems. Besides the IFs, the migration of the tropomyosins and PCNA/cyclin depended upon the buffer system and temperature utilized in the second dimension separation. Amino acid sequence-based secondary structural analysis indicates that proteins with buffer sensitive migration contain twice as much α-helix as proteins unaffected by buffer composition. Some secondary structure may persist in the α-helical rod domain of the IF monomer during two-dimensional gel electrophoresis even in the presence of sodium dodecyl sulfate and this secondary structure may be influenced by gel buffer composition. © 1991.