Abstract

Particles in solution diffract monochromatic light (i.e. lasers), and the resulting diffraction pattern is correlated to the dimensions of the particles. Because of this property, it is therefore possible to determine the accurate molecular weight, and/or level of aggregation, of purified protein samples, heterogeneous samples, and synthetic polymers, by analysing their diffraction pattern. Specifically, the requested equipment uses light scattering in two different ways: – By analyzing the temporal fluctuation of this spectrum (Dynamic Light Scattering, DLS), it is possible to identify the distribution of sizes of the particles present in a sample. This does not provide accurate molecular weight measurements, but is used as a tool to measure polymerization or aggregation, as well as for diagnostics measurement of sample homogeneity. In particular, measurements are very fast (~ 1 second), and can be performed in a high-throughput, time-resolved manner. This can be exploited for assembly inhibitor screens, or to identify conditions for stability. – When the sample is illuminated from multiple angles, the pattern of interference correlates with the radius of gyration of the particles in the sample. In turn, this can be coupled with a size-exclusion chromatography (SEC) line, which allows to separate the various molecule populations in the sample, permitting to determine their absolute molecular weight. DLS is used routinely to study protein aggregation, particularly in the context of filamentous assembly such as cytoskeletal proteins or amyloid fibrils. In particular, the possibility to use it in a high-throughput fashion has been exploited extensively for the screening of amyloid inhibitors. In contrast, SEC-MALS is mainly applied to determine the stoichiometry of macromolecular assemblies. This is particularly important for structural studies, where the determination of the exact stoichiometry is required for structure determination.