Use of excluded volume to increase the heterogeneity of pore size in agarose gels

When testing theoretical models that quantitatively describe the sieving of macromolecules during gel electrophoresis, investigators have been limited by absence of control of the heterogeneity of the size of pores in the gel. In a recent study performed by electron microscopy of thin sections (G. A. Griess et al., J. Struct. Biol. 1993, 111, 39-47), pore size heterogeneity has been increased for agarose gels by a combination of both derivatization and molecular weight reduction of the polysaccharide chains of agarose. In the present study, pore size heterogeneity is increased by a mechanism that appears to have an origin different from the origin of this previously observed increase in heterogeneity: Pore size heterogeneity is increased by addition of a polyethylene glycol (PEG) of high molecular weight (18 500) to molten agarose before gelation. In contrast, the use of a lower molecular weight PEG (either 4 000 or 7 500) causes the formation of micron-sized precipitates within a gelled network of agarose fibers. Thus far, the PEG-induced heterogeneity of pore size occurs primarily in 100-1000 μm scale zones separated from each other by interzone regions of decreased agarose fiber density. More uniform gels are needed for the study of sieving.