Factors affecting resolution, band width, number of theoretical plates, and apparent diffusion coefficients in polyacrylamide gel electrophoresis

Abstract

A systematic study of several variables affecting band width and resolution in polyacrylamide gel electrophoresis (PAGE) has been carried out. This makes it possible to determine resolution, number of theoretical plates, and an apparent diffusion coefficient in PAGE. Measurement of band position yields a linear relationship between logarithm of electrophoretic mobility and gel concentration when other variables are held constant. Similarly, measurement of band width yields a linear relationship between the logarithm of the dispersion coefficient ( D′) and gel concentration. This makes it possible to extrapolate to 0 gel concentration and to obtain as estimate of a free dispersion coefficient ( D′ 0) which is usually one or two orders of magnitude greater than the free diffusion coefficient ( D 20,w). D′ depends on protein concentration (which is a function of sample load and time), on ionic strength ( I), and on duration of electrophoresis (dependent on field strength which in turn depends on ionic strength and current). Since these several variables introduce nonlinear and interrelated correction factors, extrapolation to “infinite ionic strength,” “zero concentration,” and “infinite time” becomes difficult although it is potentially feasible at both the experimental and the theoretical level, and thus it may be possible to determine diffusion coefficients in PAGE on microgram amounts of material without the need for preliminary purification. Alternatively, PAGE in a nonsieving, anticonvectant gel at high ionic strength and for long duration may be able to provide an estimate of D 20,w. The results also support the validity of previously developed approximations for the relationship between band width and gel concentration, and for the relationship between band dispersion and electrophoretic mobility.