The sieving of spheres during agarose gel electrophoresis: Quantitation and modeling

By use of agarose gel electrophoresis, the sieving of spherical particles in agarose gels has been quantitated and modeled for spheres with a radius (R) between 13.3 and 149 mm. For quantitation, the electrophoretic mobility has been determined as a function of agarose percentage (A). Because a previously used model of sieving [D. Rodbard and A. Chrambach (1970) Proc. Natl. Acad. Sci. USA 65, 970–977] was found incompatible with some of these data, alternative models have been tested. By use of an underivatized agarose, two models, both based on the assumption of a single effective pore radius (P_E) for each A, were found to yield P_E values that were independent of R and that were in agreement with values of P_E obtained independently (P_E = 118 nm × A^−0.74): sieving by altered hydrodynamics in a cylindrical tube of radius, P_E, and sieving by steric exclusion from a circular hole of radius, P_E. The same analysis applied to a 6.5% hydroxyethylated commercial agarose yileded a steeper P_E vs A plot and also agreement of the above tow models with the data. The P_E vs A plot was significantly altered by both further hydroxyethylation and factors that cause variation in the electro‐osmosis found in commercial agarose.