Fluorometric Estimation of Viral Thermal Stability.

Differential Scanning Fluorimetry (DSF) is a rapid, economical, and a straightforward technique for estimating the thermal stability of proteins. The principle involves the binding of a fluorescent dye to thermally exposed hydrophobic pockets of a protein. The dyes used in this technique are highly fluorescent in a non-polar environment and are quenched when exposed to aqueous solution. The change in fluorescence can be used to follow unfolding of proteins induced by temperature, pH, or chaotropic agents. The method is well characterized for monomeric proteins. Here, we extend the application to supramolecular protein and nucleo-protein complexes using virus particles as an example. SYPRO-orange™ dye is the dye of choice because it is matched for use with q-PCR instruments and the fluorescence response is stable across a wide range of pH and temperatures. Advantages of this technique over standard biophysical methods include the ability for high-throughput screening of biological and technical replicates and the high sensitivity.