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