14. In normal use, the ramp rate for thermal denaturation should
be chosen carefully as a trade-off between equilibration time at
a given temperature and minimization of perturbation by
aggregation. In this case, a relatively rapid ramp rate ensures a
short experiment time, showing the utility of the method as a
rapid initial screen or check for binding. Ramp rates of this
order have been used successfully for high-throughput ligand
screening [26].
15. The software sometimes automatically inverts the sign of the
first derivative because it is primarily designed for experiments
with decreasing signal as a function of temperature. Usually,
there is a selectable option to negate this. If the software does
not calculate the first derivative for you, most common graphing software can calculate it automatically. Depending on the
degree of noise in the data, it is sometimes necessary to smooth
the data before taking the first derivative.
16. More recently, we have obtained very similar results for thermal
shift experiments monitored by DSF of the instrinsic fluorescence of HEWL, using a Nanotemper Prometheus instrument.
Using the same concentrations of HEWL and NAG3 but
omitting the SYPRO Orange dye, and running a continuous
thermal ramp from 20 to 95
C at a rate of 1
C/min in
standard capillaries, we obtained a T m of 76.2
C for HEWL,
with a ΔT m of 3
C upon addition of 1 mM NAG3.
17. You should check that this working volume is suitable for the
type of microplate that you are using in the assay.
18. Some experimentation may be required on the first attempt
with a new plate reader to identify optimal settings for the best
signal-to-noise. At the concentrations employed, the signal for
all wells containing HEWL and NAG3 should be greatly in
excess of that of the buffer with the same measurement settings. Excitation at longer wavelength (e.g., 295 nm) gives in
principle selectivity for tryptophan over tyrosine residues due
to the broader range of excitation bands for tryptophan
(advantageous in this case, since the binding site of HEWL
contains two tryptophans that contact NAG3). In practice,
when measured with a typical plate reader, this selectivity is
lost due to the wide bandwidth of excitation. Similarly, wide
bandwidth in the emission optics means that the fluorescence
intensity is effectively integrated over a broad range of wavelengths, and the exact magnitude of the signal change will
depend upon the precise properties of the plate reader optics.
One of the reasons for using a plate reader in this experiment is
to demonstrate that modern plate readers can give data of
sufficient quality for measurements of intrinsic protein fluorescence, with an acceptable trade-off of speed, sample economy,
and ease of use vs. selectivity and sensitivity.
Interactions by Multiple Methods
73
be chosen carefully as a trade-off between equilibration time at
a given temperature and minimization of perturbation by
aggregation. In this case, a relatively rapid ramp rate ensures a
short experiment time, showing the utility of the method as a
rapid initial screen or check for binding. Ramp rates of this
order have been used successfully for high-throughput ligand
screening [26].
15. The software sometimes automatically inverts the sign of the
first derivative because it is primarily designed for experiments
with decreasing signal as a function of temperature. Usually,
there is a selectable option to negate this. If the software does
not calculate the first derivative for you, most common graphing software can calculate it automatically. Depending on the
degree of noise in the data, it is sometimes necessary to smooth
the data before taking the first derivative.
16. More recently, we have obtained very similar results for thermal
shift experiments monitored by DSF of the instrinsic fluorescence of HEWL, using a Nanotemper Prometheus instrument.
Using the same concentrations of HEWL and NAG3 but
omitting the SYPRO Orange dye, and running a continuous
thermal ramp from 20 to 95
C at a rate of 1
C/min in
standard capillaries, we obtained a T m of 76.2
C for HEWL,
with a ΔT m of 3
C upon addition of 1 mM NAG3.
17. You should check that this working volume is suitable for the
type of microplate that you are using in the assay.
18. Some experimentation may be required on the first attempt
with a new plate reader to identify optimal settings for the best
signal-to-noise. At the concentrations employed, the signal for
all wells containing HEWL and NAG3 should be greatly in
excess of that of the buffer with the same measurement settings. Excitation at longer wavelength (e.g., 295 nm) gives in
principle selectivity for tryptophan over tyrosine residues due
to the broader range of excitation bands for tryptophan
(advantageous in this case, since the binding site of HEWL
contains two tryptophans that contact NAG3). In practice,
when measured with a typical plate reader, this selectivity is
lost due to the wide bandwidth of excitation. Similarly, wide
bandwidth in the emission optics means that the fluorescence
intensity is effectively integrated over a broad range of wavelengths, and the exact magnitude of the signal change will
depend upon the precise properties of the plate reader optics.
One of the reasons for using a plate reader in this experiment is
to demonstrate that modern plate readers can give data of
sufficient quality for measurements of intrinsic protein fluorescence, with an acceptable trade-off of speed, sample economy,
and ease of use vs. selectivity and sensitivity.
Interactions by Multiple Methods
73