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 form 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, KS = vimentin greater than beta-tubulin = K7 greater than K18 greater than K19 greater than 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Patton
W F
WF
Corporate Research and Development, Millipore Corporation, Bedford, Massachusetts 01730.
Chung-Welch
N
N
Lopez
M F
MF
Cambria
R P
RP
Utterback
B L
BL
Skea
W M
WM
D001888:Boric Acids; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004847:Epithelial Cells; D004848:Epithelium / Q000737:chemistry; D005069:Evaluation Studies as Topic; D005998:Glycine / Q000031:analogs & derivatives; D006801:Humans; D007381:Intermediate Filament Proteins / Q000737:chemistry*; D008970:Molecular Weight; D014325:Tromethamine
D001888:Boric Acids; D007381:Intermediate Filament Proteins; D014325:Tromethamine; C032688:boric acid; D005998:Glycine; C100184:tricine