THE BAND AREAS OF PROTEINS DETERMINED BY FLUORESCENT SCANNING IN THE COMMERCIAL AUTOMATED GEL-ELECTROPHORESIS APPARATUS

An automated gel electrophoresis apparatus, recently available commerc ially, allows one to follow the band during electrophoresis in real ti me, and lends itself therefore to an evaluation of bandwidth as a func tion of migration time (the dispersion coefficient), resolution and ba nd shape. These determinations assume the constancy of band area with migration time and at various gel concentrations. The purpose of the p resent study was to verify these assumptions. Representative proteins and sodium dodecyl sulfate (SDS)-proteins, either natively fluorescent or fluorescein carboxylate labeled, were found to exhibit band areas which approach constancy as a function of migration time in both agaro se and polyacrylamide gel electrophoresis, provided that (i) the prote in concentration under the band was low enough to obviate self-quenchi ng of fluorescence; (ii) the separation of the protein of interest fro m contaminants had progressed sufficiently during the time at which ba nd areas were measured; (iii) the baseline under the peak was sufficie ntly well defined. However, band areas decrease with increasing gel co ncentration. Protein peaks exhibited leading and trailing tails. The r atio of the combined tail area to total area appeared to be near-const ant at varying migration times. However, that ratio increases with inc reasing gel concentration. The tail area does not appear to be an arti fact of fluorometric detection since it is reproduced upon fluorimetri c analysis of the protein eluted from gel slices after electrophoresis . However, it may be due to photochemical destruction under the condit ions of repetitive fluorometric peak detection.