Protein Thermal Shift Assays Made Easy

Protein thermal shift assays enable quick and easy buffer optimization for increased protein stability. Assays can be performed with higher throughput and more buffer systems than with traditional circular dicroism detection. Bio-Rad’s family of CFX Real-Time PCR Detection Systems uses a simple protocol, described herein, to measure protein thermal stability using SYPRO Orange Fluorescent Dye. SYPRO Orange binds to hydrophobic regions in denatured proteins, illuminating the presence of unstable proteins. In the example below, we adjusted pH and buffer salt concentration in order to optimize a Tris buffer used with β-galactosidase (Aspergillus oryzae) and validated using thermal shift assays.