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How it works
DSF exploits one of the SYPRO dye's properties: that it
doesn't fluoresce in aqueous solutions, but it activates
in hydrophobic environments. In a DSF experiment, an
unfolding protein exposes hydrophobic patches that the
SYPRO dye binds to, resulting in increased fluorescence.
Once the unfolded protein starts to aggregate, the dye is
displaced and the fluorescence decreases again.
Strengths
This is an inexpensive method that is relatively easy to use
and is suitable for high-throughput screening.
Weaknesses
This method harbors several disadvantages. First, only a
limited amount of thermal stability information is collected
(Typicallyt only one Tm is determined), so it's not an ideal
approach for large proteins such as monoclonal antibodies
that have multiple Tm. Also, because the SYPRO dye
interacts directly with the protein, it likely alters its
unfolding behavior by promoting transitions into the
unfolded state. In general, scientists who are looking to
mimic physiological conditions as close as possible prefer
to use label-free methods to limit the potential confounding
influence of fluorescent probe binding on protein
properties. In addition, DSF is incompatible with a number
of common excipients, especially surfactants such as PS20
and similar vehicles.
Conclusion
DSF/thermofluor is an inexpensive, easy-to-use method
for the high-throughput screening of small molecule
interactions, but because of its limitations in detecting
multiple unfolding events and large proteins, it's not
the ideal choice for many large molecule biologics
development workflows.
Differential scanning fluorimetry (DSF) is a general description for a group of thermal stability assays that use a
solvatochromic dye, or a dye that changes color due to a change in solvent polarity (SYPRO Orange is the most commonly
used dye). Assays are typically performed using a qPCR instrument, a tool that is present in many molecular biology
research facilities. Note that most people prefer to call the technique by its original trademark name, Thermofluor.
Differential Scanning Fluorimetry
