Determination of Hydrodynamic Radius of Proteins by Size Exclusion Chromatography

Size exclusion chromatography (SEC) or gel filtration is a hydrodynamic technique that separates molecules in solution as a function of their size and shape. In the case of proteins, the hydrodynamic value that can be experimentally derived is the Stokes radius (R_s), which is the radius of a sphere with the same hydrodynamic properties (i.e., frictional coefficient) as the biomolecule. Determination of R_s by SEC has been widely used to monitor conformational changes induced by the binding of calcium (Ca²⁺) to many Ca²⁺-sensor proteins. For this class of proteins, SEC separation is based not just on the variation in protein size following Ca²⁺ binding, but likely arises from changes in the hydration shell structure. This protocol aims to describe a gel filtration experiment on a prepacked column using a Fast Protein Liquid Chromatography (FPLC) system to determine the R_s of proteins with some indications that are specific for Ca²⁺ sensor proteins.