The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability

Nat Protoc. 2007;2(9):2212-21. doi: 10.1038/nprot.2007.321.


Differential scanning fluorimetry (DSF) is a rapid and inexpensive screening method to identify low-molecular-weight ligands that bind and stabilize purified proteins. The temperature at which a protein unfolds is measured by an increase in the fluorescence of a dye with affinity for hydrophobic parts of the protein, which are exposed as the protein unfolds. A simple fitting procedure allows quick calculation of the transition midpoint; the difference in the temperature of this midpoint in the presence and absence of ligand is related to the binding affinity of the small molecule, which can be a low-molecular-weight compound, a peptide or a nucleic acid. DSF is best performed using a conventional real-time PCR instrument. Ligand solutions from a storage plate are added to a solution of protein and dye, distributed into the wells of the PCR plate and fluorescence intensity measured as the temperature is raised gradually. Results can be obtained in a single day.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Citrate (si)-Synthase / chemistry
  • Citrate (si)-Synthase / metabolism
  • Enzyme Stability
  • Fluorescent Dyes / analysis
  • Fluorometry / methods*
  • Ligands
  • Oxaloacetic Acid / metabolism
  • Polymerase Chain Reaction
  • Protein Conformation*
  • Protein Denaturation*
  • Protein Folding
  • Swine / metabolism
  • Temperature


  • Fluorescent Dyes
  • Ligands
  • Oxaloacetic Acid
  • Citrate (si)-Synthase