Calibration of size-exclusion chromatography: use of a double Gaussian distribution function to describe pore sizes.

A method for the calibration of size-exclusion chromatographic columns is proposed that takes into account the nonlinear dependence of the Stokes radius Rs upon the partition coefficient KD. The method is based on the assumption that the pore size distribution of a molecular sieve column can be described by the sum of two Gaussian distributions and has been successfully tested with low-pressure chromatography gels (Sephacryl and Superose) and HPLC gels (TSK SW) over a wide range of protein sizes. An application of this method is described, in which aggregation states of the membrane protein prostaglandin H2 synthase solubilized in nonionic detergents are estimated.