Multi-angle light scattering and dynamic light scattering coupled to size exclusion chromatography

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Experiments in protein biochemistry and structural biology entirely rely on the availability of purified proteins in isolation or in complex with other proteins, RNA, DNA or lipids. Protein particle solutions are characterized by a number of different parameters like particle concentration, hydrodynamic radius and molar mass. The determination of these parameters and thus the indispensable information about the homogeneity of protein(complex) particles in solution is of utmost importance for understanding principles of their assembly and/or biogenesis and molecular functions. Furthermore, many downstream methods like crystal structure determination or biochemical and biophysical approaches such as in vitro-reconstitution experiments either depend on or at least benefit from such information.With this proposal, we apply for a setup consisting of a combination of multi-angle light scattering (determination of the absolute molar mass), dynamic light scattering (determination of hydrodynamic radius) and a refractive index detector (absorption independent concentration determination) coupled to size exclusion chromatography in a chromatography refrigerator. This setup will be used by several independent institutes and research groups at the Ulm University working with proteins or protein complexes, ensuring efficient and maximum utilization of the setup.The equipment will be used to determine the absolute molar mass as well as its distribution and hydrodynamic radii of native and unlabeled biological particles in solution. These particles may be proteins and protein-conjugate complexes (the conjugates being carbohydrates, nucleic acids or lipids). This information will help us to understand the molecular functions of biomolecules either in their healthy or diseased states or when used for therapeutic approaches. In addition, it also serves as tool for quality control in a number of projects and will significantly increase the chances of crystallization of proteins and their complexes. Since no comparable equipment is available on the entire campus, this equipment will considerably increase the analytical possibilities for biological samples at the Ulm University, including research conducted as part of three protein-centered, DFG-funded research networks on campus.