Determination of protein-ligand interactions using differential scanning fluorimetry.
Journal of Visualized Experiments
Journal of Visualized Experiments (JoVE)
Copyright © 2014 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License
A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.
This work was funded by grant from the BBSRC (grant number BB/H019685/1 and BB/E527663/1) to the University of Exeter.
Research Support, Non-U.S. Gov't
This is the final version of the article. Available from JoVE via the DOI in this record.
Vol. 91, e51809
Place of publication