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

Abstract

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 nm. 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 (PE) for each A, were found to yield PE values that were independent of R and that were in agreement with values of PE obtained independently (PE = 118 nm X A-0.74): sieving by altered hydrodynamics in a cylindrical tube of radius, PE, and sieving by steric exclusion from a circular hole of radius, PE. The same analysis applied to a 6.5% hydroxyethylated commercial agarose yielded a steeper PE vs A plot and also agreement of the above two models with the data. The PE vs A plot was significantly altered by both further hydroxyethylation and factors that cause variation in the electro-osmosis found in commercial agarose.

Interfacing solid‐state nanopores with gel media to slow DNA translocations

“…One large problem associated with determining these gel pore sizes is that each method of measurement tends to give a different reading, due to the inherent sampling bias of each different method [83,87]. For instance, determining the gel pore size in 1% agarose using the mobility of latex spheres results in an estimation of the diameter of 150 nm [88], while the use of an atomic force microscope yields greater than 400 nm in diameter [89].…”

Gels for the Propagation of Bacteriophages and the Characterization of Bacteriophage Assembly Intermediates

“…The turbidity of an agarose gel decreases as (1) the temperature of gelation and buffer ionic strength decrease, and (2) the agarose molecular weight increases (Griess et al, 1993;Griess et al, 1998;Serwer & Griess, 1999), with an associated decrease in the radius of the effective pore (P E ). If one adds consideration of the agarose source-, purification-and derivatization-dependence of P E (Griess et al, 1989;Griess et al, 1998), one can only conclude that P E -dependent results from different studies cannot be compared quantitatively unless one is willing to tolerate the likelihood of P E errors of at least 100%. In general, quantitative comparisons should be performed with internal standards.…”

“…Agarose is a sub-fraction of agar that has a relatively low density of charged groups (reviewed in Rees, 1972). The extent of residual charge is often used to name agarose preparations via the field-induced flow of buffer that gel-attached charged groups cause (electro-osmosis, abbreviated EEO; Griess et al, 1989). The minimum agarose concentration for gel formation varies somewhat with agarose EEO, and agarose chain length but can be as low as 0.03% for a high-strength agarose, when the gel is supported at its sides by embedding in a more concentrated gel (Serwer et al, 1988).…”

“…One pays for the increase in sieving-based resolution with an increase in the time of fractionation. A quantitative analysis of these effects for spheres is in Griess et al (1989).…”

“…The culmination of a series of sieving-based measurements of P E produced the following equation that describes the relationship between P E (in nm) and the percentage, A, of the LE (low EEO) agarose usually used; gels were formed at ~25 °C in buffer that contained 0.025 M sodium phosphate, pH 7.4, 0.001 M MgCl 2 (Griess et al, 1989). P E = 148A -0.87 .…”