Application note: Using SYPRO^® Orange to Detect Protein Melting Temps

Investigation of thermal protein stability in high throughput using the application of thermal shift assay using SYPRO ^® Orange in 96 well format.

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.