UNIT 29.5 HPLC-SEC Characterization of Membrane Protein-Detergent Complexes

Abstract

Determination of the oligomeric state of integral membrane proteins in detergent solutions is a challenging task because the amount of detergent associated with the protein is typically unknown and unpredictable. Methods that estimate the molecular weight of proteins from their hydrodynamic properties in solution are not suitable for detergent-solubilized membrane proteins. However, size-exclusion chromatography (SEC) performed in combination with analyses of static light scattering (SLS), ultraviolet absorbance (UV), and refractive index (RI) provides a universal method for determination of the molar masses of biopolymers and protein-detergent complexes. The light scattered by a protein is directly proportional to its molecular mass, irrespective of shape, and any additional contributions due to bound detergent molecules can be quantitatively accounted for by the additional combined analysis of ultraviolet absorbance and refractive index information. The primary intention of this unit is to describe how to apply the combination of high-performance liquid chromatography SEC and SLS-UV-RI to evaluate molecular mass and the physicochemical heterogeneity of purified membrane protein-detergent complexes. Curr. Protoc. Protein Sci. 68:29.5.1-29.5.12. © 2012 by John Wiley & Sons, Inc.