Estimation of the molecular weights of proteins by Sephadex gel-filtration

Abstract

When a mixture of molecules is passed through a column of porous gel granules, the molecules may appear in the effluent in order of decreasing size. Fractionation is believed to occur when diffusion of the molecules into the gel pores is restricted but not prevented because of their size, and because they pass through the column at rates that are related inversely to the fluid volume accessible to them within the column. Lathe & Ruthven (1956) suggested that the dimensions of protein and poly- saccharide molecules might be estimated by this technique, now widely known as gel-filtration. However, evaluation of their suggestion is difficult because of uncertainty about the size and shape of macromolecules in solution (Schachman, 1960). Although molecular weight is unlikely to be a good approximation for size in comparing such dis- similar molecules as proteins and polysaccharides, the correlation between molecular weights and gelfiltration behaviour of dextrans (Granath & Flodin, 1961) indicates that, for a homogeneous series of macromolecules, size and molecular weight are closely related. Andrews (1962) obtained evidence from experiments with agar-gel columns that this was also true for a number of proteins, and showed that gel-filtration can be used as a comparative method to give useful estimates of the molecular weights of proteins.