Analysis of Membrane Protein by Multi-Detector SEC
Work performed in conjunction with the Membrane Protein Laboratory, Imperial College, London
Introduction
Size-exclusion chromatography (SEC) is a tool that is commonly used in the biological sciences. Typically, it is used to measure the molecular weight of an unknown sample by referencing the elution time of the unknown against that of standards of known molecular weight. This can be done with a single concentration detector such as UV or refractive index (RI); but to make the measurement accurate, additional detectors must be used. Advanced multi-detector SEC systems now include up to four detectors including UV, RI, light scattering and intrinsic viscosity. Light scattering detectors allow the measurement of molecular weight without the need for column calibration. Intrinsic viscosity is a measure of molecular density and enables structural changes to be assessed. A combination of light scattering and intrinsic viscosity allows size (Rh) to be calculated.
Maintaining the detectors in series is essential for maintaining maximum sensitivity across all four detectors.
Studies of membrane proteins are becoming increasingly frequent in molecular biology research. Crystallization is one of the main goals of this research in order to characterize the structure of a protein; however, crystallization of these proteins has proven difficult. Crystallization of a membrane protein will depend on many factors such as the protein purity and the detergent concentration or type. Removal of too much of the detergent component of a membrane protein complex can lead to degradation of the protein and reduce the likelihood of crystallization. Characterizing and optimizing the proportion of protein and detergent in a purified membrane protein sample can provide valuable insight into the likelihood of crystallization and the protein content of the protein detergent complex (PDC).
In this application note a purified membrane protein and a bacterial membrane protein involved in multidrug resistance, is characterized using the Viscotek TDAmax system, a complete size-exclusion chromatography system with refractive index (RI), ultraviolet (UV), light scattering (LS) and intrinsic viscosity (IV) detectors.
