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 Microarray plates as a convenient ligand library, although the methods described should be generally applicable to other library formats as well.
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Acknowledgment
The authors gratefully acknowledge financial support from a Marsden Fast Start grant administered by the Royal Society of New Zealand. MKGE is supported by a University of Otago Doctoral Scholarship.
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Ehrhardt, M.K.G., Warring, S.L., Gerth, M.L. (2018). Screening Chemoreceptor–Ligand Interactions by High-Throughput Thermal-Shift Assays. In: Manson, M. (eds) Bacterial Chemosensing. Methods in Molecular Biology, vol 1729. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7577-8_22
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DOI: https://doi.org/10.1007/978-1-4939-7577-8_22
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