Protein Crystallography and Biophysics Centre (BiophysX)
Institute of Structural and Molecular Biology (ISMB)
Birkbeck College / University College London

Thermal Shift Assays (Thermofluor)
Biorad MyIQ5

Thermal Shift Assays (or known as Thermofluor) measure the thermal denaturation of proteins using fluorescence. The method uses SyproOrange as dye that fluoresces in a hydrophobic environment. It binds to a protein’s hydrophobic areas that get exposed upon unfolding, so fluorescence increases when the protein fold melts. It is not a highly accurate biophysical method, but it is performed in 96-well format and convenient to screen different solution conditions or ligands all at once in a small volume.

The current instrument is not suitable for membrane proteins and in general with samples that have large hydrophobic exposed areas.

Set up and Sample Requirements

  • Sample Volume (per well): 25 μl
  • Sample Concentration (per well): for a 20-30kDa protein 5 μΜ or about 0.1-0.2 mg/ml of protein works well. 
  • Dye concentration: a 5x final concentration is a good start, can be 2.5x - 10x.
  • Optional positive control: Lysozyme 5 mg/ml in 19 mM glycine pH 2
  • Triplicates for each sample is strongly recommended.
Ericsson et al, Analytical Biochemistry, 354, 289-298 (2006)
Niesen et al, Nature Protocols, 2(9), 2212-2221, (2007)

Please contact us for detailed protocols and planning your experiment.

ISMB Protein Crystallography and Biophysics Centre, Birkbeck, University of London
Last modified April 2021