Protein Bandwidth in Gel Electrophoresis: A Primary Function of Migration Distance

Abstract

By use of the fluorescent protein R-phycoerythrin, a number of factors have been investigated as potential sources of band broadening in agarose and polyacrylamide gels: temperature gradients, Joule heating, conductivity differences between analyte and buffer, electroendosmosis, microheterogeneity due to charge density differences, etc. It was found that none of these factors could be the major source of band broadening. Recently, it has been shown that diffusion is also not the predominant source of R-phycoerythrin band spreading in agarose gels and that the bandwidth depends linearly on migration distance and time (Yarmola, E.; Calabrese, P. P.; Chrambach, A.; Weiss, G. H. J. Phys. Chem. 1996, 101, 2381). The present data collected in agarose and polyacrylamide gels confirm the linear dependence of bandwidth on migration distance and describe the slope of this dependence as a function of electrophoretic conditions. Among all the factors studied that may impact on band spreading, only the interaction with the gel and microheterogeneity cannot be ruled out. This conclusion with regard to the mechanisms of band spreading agrees with that obtained recently by capillary electrophoresis in polymer solutions (Radko, S. P.; Weiss, G. H.; Chrambach, A. J. Chromatogr. A 1997, 781, 277−286).