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Thermal Shift Assay for Characterizing the Stability of RNA Helicases and Their Interaction with Ligands

DOI https://doi.org/10.1007/978-1-0716-0935-4_5

Scientific Field Life & Health Sciences

Fellow Dr Emmanuel Saridakis
Methods in Molecular Biology, 2209, pp. 73-85. Springer Nature, 2020

Saridakis Emmanuel1, 2, 3, Coste Franck3

1LE STUDIUM Institute for Advanced Studies, 45000 Orléans, France

2Laboratory of Structural and Supramolecular ChemistryInstitute of Nanoscience and Nanotechnology, National Centre for Scientific Research “DEMOKRITOS”, Athens, Greece

3Centre de Biophysique Moléculaire, CNRS UPR4301Orléans Cedex 2France

ABSTRACT

Thermofluor or thermal shift assay is an easily implementable, high-throughput method for assessing the thermostability of proteins and the influence of various ligands on that stability. It is particularly useful for the assaying of ligands that may stabilize oligomeric helicases, which rely on both substrates (oligonucleotides) and nucleotide cofactors (ATP analogues) for their stability in a functional state. In this chapter, we describe the rationale and present a basic protocol for the use of this technique. Multi-ligand screening is also discussed via a worked example of the stabilization of a hexameric RNA helicase, a target protein for structural studies in our laboratories.


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Methods in Molecular Biology