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 ), which is the radius of a sphere with the same hydrodynamic properties ( , frictional coefficient) as the biomolecule. Determination of R by SEC has been widely used to monitor conformational changes induced by the binding of calcium (Ca ) to many Ca -sensor proteins. For this class of proteins, SEC separation is based not just on the variation in protein size following Ca 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 of proteins with some indications that are specific for Ca sensor proteins.