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

  • Maximilian K. G. Ehrhardt
  • Suzanne L. Warring
  • Monica L. Gerth
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1729)

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.

Keywords

Ligand Signal molecule Screening Binding Differential scanning fluorometry Fluorescence thermal shift assay Chemoreceptor Ligand binding domain High-throughput 

Notes

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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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Maximilian K. G. Ehrhardt
    • 1
  • Suzanne L. Warring
    • 1
  • Monica L. Gerth
    • 2
  1. 1.Department of BiochemistryUniversity of OtagoDunedinNew Zealand
  2. 2.Department of Biological SciencesVictoria University of WellingtonWellingtonNew Zealand

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