Cutting edge biophysical tools to determine biomolecular interactions and characterize protein stability in solution
published: July 9, 2018, recorded: May 2018, views: 405
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Introduction to Micro Scale Thermophoresis Analysis of bio-molecular interactions, such as protein-protein, protein-nucleic acid or protein-small molecule, not only helps to develop therapeutics or diagnostics techniques, but it also provides important insights into cellular processes. Here we present MicroScale Thermophoresis (MST) for the investigation of affinities of biomolecular interactions. MST is a biophysical technique that measures the strength of the interaction between two molecules by detecting variations in fluorescence signal as a result of an IR-laser induced temperature change. The range of the variation in the fluorescence signal correlates with the binding of a ligand to the fluorescent target Introduction to nanoDSF The fluorescence of tryptophans in a protein is strongly dependent on its close surroundings. By following changes in fluorescence, chemical and thermal stability can be assessed in a truly label-free fashion. The dual-UV technology by NanoTemper allows for rapid protein unfolding analysis, providing an unmatched scanning speed and data point density. This yields ultra-high resolution melting profiles of proteins which allow for detection of even minute unfolding signals. Furthermore, since no secondary reporter fluorophores are required, protein solutions can be analyzed independent of buffer compositions. In addition, information on protein aggregation can be recorded in parallel, providing insight into colloidal stability of the sample. The presentation will cover biophysical concepts of the techniques showing benefits of MST and nanoDSF technologies platforms, and will be followed by specific examples of applications towards various experimental systems.
Download slides: biophysics2018_nowak_biophysical_tools_01.pdf (4.3 MB)
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