Fluorescence-based thermal shift assays.

Thermal shift assays are one of the label-free technologies gaining popularity in drug discovery and academic research. This review summarizes the various detection formats used in these assays, and provides an in-depth presentation of fluorescence-based thermal shift assays. The dual applications of stability profiling and affinity ranking are highlighted, and basic experimental protocol and data analysis algorithms are recommended. The advantages and limitations of the technology are also discussed to provide guidance for meaningful data interpretation. In addition, an emerging paradigm is described that incorporates thermal shift assays with other label-free biosensors and conventional IC50 assays to derive a mechanism of action-informed SAR in lead identification and optimization during drug discovery.