Thermal Shift Assay using SYPRO Orange to Detect Protein Melting Temperatures

The stability of proteins depends on ligand interactions, buffer conditions or changes in conformation, which is traditionally investigated by time-consuming Circular Dichroism (CD) Spectroscopy. In addition, the thermal shift assay is based on temperature-induced denaturation and can be monitored using SYPRO Orange. This fluorescence dye is a naturally quenched dye and interacts with the hydrophobic core of proteins which is exposed after denaturation.
Through this interaction, the SYPRO Orange dye releases a fluorescence signal. The performance of a melting curve by using the qTOWER3 real-time PCR thermal cycler thus allows an easy and fast determination of protein melting temperatures. The midpoint or melt peak of the generated melting curve corresponds to the melting temperature (Tm value) of the protein under current conditions. Protein thermal shift assays are sensitive and rapid tools to examine protein thermal stability, helping to evaluate proteinligand binding and thus to find optimal buffer conditions or analyze protein variations. In this experiment, the Tm values of α-Chymotrypsinogen A in TBS with three different NaCl concentrations were determined.

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