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In Thermofluor technique, SYPRO Orange, an environment-sensitive dye, is used to measure the unfolding of the protein as the temperature rises. SYPRO® Orange partitions itself into the melted protein and hence the overall effect is an increase in fluorescence with increasing temperature. With this method, Tm (melting temperature) is estimated, which is a measure of mid-point of a thermal unfolding curve. Unfolding does not depend upon neighboring proteins. When a protein starts to unfold, SYPRO® Orange binds to the protein prematurely and causes it to unfold further through a phenomenon known as “the wedge effect”.
On the other hand, the ProteoStat® thermal shift stability assay uses a molecular rotor dye that binds to the quaternary structure of protein aggregates. As proteins unfolds, they subsequently aggregate. Aggregation depends upon neighboring proteins. The higher the protein concentration, the greater is the tendency to aggregate. While SYPRO® Orange dye measures Tm value, ProteoStat® dye measures Tagg (aggregation temperature), which is the midpoint of a thermal aggregation curve. In this method, the increase in fluorescence intensity readout is directly proportional to the amount of aggregated protein.