used, but the wavelength range must be adapted (250–340 nm)
and greater protein concentration and/or path lengths are usually
used to compensate for the smaller CD signal (and taking advantage of the lower molar absorbance) in this region.
2.3.2 DSF of Intrinsic
Fluorophores
DSF of intrinsic tryptophan fluorescence is typically carried out on a
dedicated instrument (such as the Prometheus—Nanotemper
Technologies where samples are held and heated in capillaries). A
backscattering measurement is also performed simultaneously to
detect aggregation that may occur (before or after denaturation).
DSF with intrinsic fluorophores can be effective with membrane
proteins; however, in interpreting results, bear in mind that the
detergent or amphipols used to solubilize them may disturb the
330-nm to 350-nm frequency shift upon which the method relies.
A simple protocol for buffer screening via DSF is presented
below:
1. Switch on the instrument and let the system warm up for at
least 15 min.
2. Prepare a stock solution of the target protein with an absorbance at 280 nm of ~1 AU.
3. Mix 1μL of protein with 10μL of each buffer to be screened. If
available, preparation of buffers in multiwell plates with a robot
or the use of commercial buffer-screening kits is recommended
to avoid mistakes and to increase reproducibility.
4. Repeat the procedure for all buffer conditions to be tested.
5. Fill the capillaries with the sample solutions and put them into
the DSF instrument.
6. Adjust the excitation power so that the fluorescence signal is
higher than 2000 counts to get good signal-to-noise. If the
signal remains lower than 2000 counts, increase the initial
concentration of the protein.
7. Set the temperature range from 20 to 95
C and select a
temperature gradient. The choice of the temperature gradient
is important as it is linked to the unfolding activation energy via
the Arrhenius equation. Typically, a gradient of 0.5
C/min
is used.
8. Start the measurement.
Automated analysis provides T m as the temperature of the
maximum rate of change in the emission ratio. Visual inspection
of the ratio vs. temperature plots should always be used to provided
assurance that a simple two-state model of the unfolding process is
appropriate. Changes in buffer can also lead to more complex
changes in the plots (i.e., not only simple shifts of T m ) such as a
transition from a complex multitransition trace (perhaps explicable
36
Bertrand Raynal et al.
and greater protein concentration and/or path lengths are usually
used to compensate for the smaller CD signal (and taking advantage of the lower molar absorbance) in this region.
2.3.2 DSF of Intrinsic
Fluorophores
DSF of intrinsic tryptophan fluorescence is typically carried out on a
dedicated instrument (such as the Prometheus—Nanotemper
Technologies where samples are held and heated in capillaries). A
backscattering measurement is also performed simultaneously to
detect aggregation that may occur (before or after denaturation).
DSF with intrinsic fluorophores can be effective with membrane
proteins; however, in interpreting results, bear in mind that the
detergent or amphipols used to solubilize them may disturb the
330-nm to 350-nm frequency shift upon which the method relies.
A simple protocol for buffer screening via DSF is presented
below:
1. Switch on the instrument and let the system warm up for at
least 15 min.
2. Prepare a stock solution of the target protein with an absorbance at 280 nm of ~1 AU.
3. Mix 1μL of protein with 10μL of each buffer to be screened. If
available, preparation of buffers in multiwell plates with a robot
or the use of commercial buffer-screening kits is recommended
to avoid mistakes and to increase reproducibility.
4. Repeat the procedure for all buffer conditions to be tested.
5. Fill the capillaries with the sample solutions and put them into
the DSF instrument.
6. Adjust the excitation power so that the fluorescence signal is
higher than 2000 counts to get good signal-to-noise. If the
signal remains lower than 2000 counts, increase the initial
concentration of the protein.
7. Set the temperature range from 20 to 95
C and select a
temperature gradient. The choice of the temperature gradient
is important as it is linked to the unfolding activation energy via
the Arrhenius equation. Typically, a gradient of 0.5
C/min
is used.
8. Start the measurement.
Automated analysis provides T m as the temperature of the
maximum rate of change in the emission ratio. Visual inspection
of the ratio vs. temperature plots should always be used to provided
assurance that a simple two-state model of the unfolding process is
appropriate. Changes in buffer can also lead to more complex
changes in the plots (i.e., not only simple shifts of T m ) such as a
transition from a complex multitransition trace (perhaps explicable
36
Bertrand Raynal et al.