3.4.2 Nano Differential
Scanning Fluorimetry
(nanoDSF)
A simple protocol to run a nanoDSF experiment with a MAST2PDZ solution free or in complex with a peptide on the Prometheus
NT.48 is given below:
1. Turn on the instrument and allow the system to warm up for at
least 15 min.
2. Clean the system with 70% ethanol.
3. Prepare in four different PCR tubes: 10 μl of buffer, 10 μl of
PDZ sample, 10 μl of PDZ sample with peptide a tenfold
concentration excess and 10 μl of a tenfold more concentrated
peptide solution.
4. Insert a capillary into the tubes and position them in order on
the rack in the DSF instrument.
5. Adjust the excitation power so that the fluorescence signal is
greater than 2000 counts to obtain clear denaturation
transitions.
6. Set the temperature range from 20 to 95
C with a desired
temperature gradient of (between 0.1 and 5
C).
7. Perform all measurements with the same temperature ramp.
8. Start the measurement.
3.4.3 Differential
Scanning
Calorimetry (DSC)
DSC is useful to characterize the stability of a protein by measuring
the emission or absorption of heat from a biomolecule during a
controlled increase or decrease in temperature. This allows the
study of molecules in a native state and is widely applied in protein
engineering, for rational drug design and biopharmaceutical production, when the development of stable proteins is a critical goal.
The thermal core of a DSC system consists of two cells, a
reference and a sample cell maintained at the same temperature,
as they are heated. To perform a DSC measurement, the reference
cell is first filled with buffer and the sample cell with the sample
solution. These cells are then heated at a constant scan rate. The
absorption of heat that occurs when a protein unfolds causes a
temperature difference (ΔT) between the cells, resulting in a thermal gradient across the Peltier units. From the ΔT values, DSC
profiles are generated to provide information on thermal stability
by measuring the thermal transition temperature (thermal transition midpoint or melting temperature, T m ) and the energy required
to break the stabilization interactions of the tertiary structure
(enthalpy) of proteins. Comparisons are made between samples
(e.g., wild-type and mutant proteins or various conditions of
buffer) and differences in the derived values indicate differences in
thermal stability and structural conformation. Compared with
other methods of evaluating the thermal stability of protein conformations, DSC is cost-effective and only requires few sample
preparation steps.
116
Ce ´ lia Caillet-Saguy et al.
Scanning Fluorimetry
(nanoDSF)
A simple protocol to run a nanoDSF experiment with a MAST2PDZ solution free or in complex with a peptide on the Prometheus
NT.48 is given below:
1. Turn on the instrument and allow the system to warm up for at
least 15 min.
2. Clean the system with 70% ethanol.
3. Prepare in four different PCR tubes: 10 μl of buffer, 10 μl of
PDZ sample, 10 μl of PDZ sample with peptide a tenfold
concentration excess and 10 μl of a tenfold more concentrated
peptide solution.
4. Insert a capillary into the tubes and position them in order on
the rack in the DSF instrument.
5. Adjust the excitation power so that the fluorescence signal is
greater than 2000 counts to obtain clear denaturation
transitions.
6. Set the temperature range from 20 to 95
C with a desired
temperature gradient of (between 0.1 and 5
C).
7. Perform all measurements with the same temperature ramp.
8. Start the measurement.
3.4.3 Differential
Scanning
Calorimetry (DSC)
DSC is useful to characterize the stability of a protein by measuring
the emission or absorption of heat from a biomolecule during a
controlled increase or decrease in temperature. This allows the
study of molecules in a native state and is widely applied in protein
engineering, for rational drug design and biopharmaceutical production, when the development of stable proteins is a critical goal.
The thermal core of a DSC system consists of two cells, a
reference and a sample cell maintained at the same temperature,
as they are heated. To perform a DSC measurement, the reference
cell is first filled with buffer and the sample cell with the sample
solution. These cells are then heated at a constant scan rate. The
absorption of heat that occurs when a protein unfolds causes a
temperature difference (ΔT) between the cells, resulting in a thermal gradient across the Peltier units. From the ΔT values, DSC
profiles are generated to provide information on thermal stability
by measuring the thermal transition temperature (thermal transition midpoint or melting temperature, T m ) and the energy required
to break the stabilization interactions of the tertiary structure
(enthalpy) of proteins. Comparisons are made between samples
(e.g., wild-type and mutant proteins or various conditions of
buffer) and differences in the derived values indicate differences in
thermal stability and structural conformation. Compared with
other methods of evaluating the thermal stability of protein conformations, DSC is cost-effective and only requires few sample
preparation steps.
116
Ce ´ lia Caillet-Saguy et al.