Screening Chemoreceptor-Ligand Interactions by High-Throughput Thermal-Shift Assays.

Abstract

Identifying the ligands sensed by chemoreceptors remains challenging, in part because current screening methods are low-throughput, costly, and/or time-consuming. In contrast, fluorescence thermal shift (FTS) assays provide a fast and inexpensive approach to chemoreceptor-ligand screening. In FTS assays, the temperature at which a protein denatures is measured by monitoring the fluorescence of a dye with affinity for hydrophobic regions of the protein, which are exposed as the protein unfolds. A detectable increase (or "shift") in the melting temperature (T m ) of the protein in the presence of a potential ligand indicates binding. Here, we present our protocol for using FTS assays for the screening of chemoreceptor ligands in a high-throughput, 96-well plate format. We have also included details on the use of Biolog Phenotype Microarrayplates as a convenient ligand library, although the methods described should be generally applicable to other library formats as well.