Chapter 23 Gel Filtration

Abstract

Publisher Summary The chapter describes the gel filtration technique. Among the chromatographic techniques employed for protein purification, gel filtration is unique in the sense that fractionation is based on the relative size of protein molecules. In contrast to conventional filtration, none of the proteins is retained by a gel-filtration column. This feature is, at the same time both, a strength and a weakness of gel filtration. It is a strength because the function of fragile proteins is not damaged by binding to a chromatographic support, and it is a weakness because the absence of such binding limits the resolution of the chromatography. Gel filtration is performed using porous beads as the chromatographic support. A column constructed from such beads will have two measurable liquid volumes, the external volume that consists of the liquid between the beads, and the internal volume that consists of the liquid within the pores of the beads. Large molecules will equilibrate only with the external volume while small molecules will equilibrate with both the external and internal volumes. A mixture of proteins is applied in a discrete volume or zone at the top of a gel-filtration column and is allowed to percolate through the column. The large protein molecules are excluded from the internal volume and they first emerge from the column. The smaller protein molecules, which can access the internal volume, emerge later. The dimensions important to gel filtration are the diameter of the pores that access the internal volume and the hydrodynamic diameter of the protein molecules.