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.
Our new CFX-DUET has a much higher
sensitivity and dynamic range and it
is also suitable for several
additional applications offering a
wide range of
excitation/emission wavelengths.
CFX
Duet Real-Time
PCR System
Protein thermal shift assays
Gene expression without
requiring ROX
End-point analysis for
sequence detection
Protein thermal shift assays
Assay optimization with
thermal gradient
Genotyping
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 for protein
stability assays
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.
Biorad MyIQ5
The old
instrument is still available
for use as back-up
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.
