Light scattering techniques are widely used in protein characterization. Dynamic light scattering (DLS) is established in the measurement of particle and molecular size, and in studying the interactions between proteins. Electrophoretic light scattering (ELS) is used to measure the electrophoretic mobility of particles in dispersion or molecules (such as proteins) in solution. This mobility is often converted into zeta potential to enable comparison of materials under different conditions. In the case of proteins, the measurement of protein mobility allows the calculation of protein charge, which in turn relates to factors such as activity and reaction kinetics. Recent advances in instrumentation and methodologies are addressing the technical challenges of using light scattering to make mobility measurements on proteins.
Experimentally, protein mobility measurements present two practical challenges. First, working with protein solutions often means working with dilute concentrations, low DLS count rates, and low electrophoretic mobilities. Second, the act of applying an electric field to the sample can damage the protein by stimulating aggregation, with resultant mobility measurements reflecting the aggregate molecules rather than the native protein.
A new approach combines a high sensitivity light scattering system (Zetasizer Nano ZSP, Malvern Instruments) with a diffusion barrier technique that separates the molecules in the sample from the electrodes, to avoid the risk of aggregation. A measurement protocol regulates voltage and temperature; and automatic size measurements before and after the electrophoretic mobility measurement verify that no aggregation has occurred.