Applications of Differential Scanning Fluorometry and Related Technologies in Characterization of Protein–Ligand Interactions
Differential scanning fluorometry (DSF) is an efficient and high-throughput method to analyze protein stability, as well as detect ligand interactions through perturbations of the protein’s melting temperature. The method monitors protein unfolding by observing the fluorescence changes of a sample, whether through an environmentally sensitive fluorophore or by intrinsic protein fluorescence, while a temperature gradient is applied. Here, we describe in detail how to develop and optimize DSF assays to identify protein–ligand interactions while exploring different buffer and additive conditions. Analysis of the data and further applications of the method are also discussed.
Key wordsDifferential scanning fluorometry DSF Thermal shift assay Thermal denaturation Protein stability Label-free DSF NanoDSF Protein–ligand interaction
- 10.Moggridge J, Biggar K, Dawson N, Storey KB (2017) Sensitive detection of immunoglobulin G stability using in real-time isothermal differential scanning fluorimetry: determinants of protein stability for antibody-based therapeutics. Technol Cancer Res Treat. doi: https://doi.org/10.1177/1533034617714149.CrossRefGoogle Scholar
- 11.Svilenov H, Markoja U, Winter G (2018) Isothermal chemical denaturation as a complementary tool to overcome limitations of thermal differential scanning fluorimetry in predicting physical stability of protein formulations. Eur J Pharm Biopharm 125:106–113. https://doi.org/10.1016/j.ejpb.2018.01.004CrossRefPubMedGoogle Scholar