be used here. In SPR and other surface-based sensor technologies, the ligand is designated as the binding component
immobilized on the sensor surface (irrespective of size) while
the other partner in the interaction is then termed the analyte.
3. If measurements are being made in solvent with a significantly
different heat capacity, e.g., buffers containing high salt concentration, organic solvents such as DMSO, or cryo preservatives such as glycerol, then the reference cell should be filled
with the same solvent.
4. Heat is being applied to both ITC cells during measurement
with a very small excess positive differential power being
applied to the sample cell. Since the cells are well insulated in
the ITC instrument, this energy input leads to a very small
increase in the absolute temperature of the cells over the duration of the measurement. However, this is very far from a level
significant for measurement accuracy and probably smaller
than temperature variations seen in many other techniques.
The temperature of measurement reported should be that set
in the instrument control software and used by the ITC during
its initial equilibration phase.
5. The integration of the excess peaks in differential power seen in
ITC data by defining baseline regions before and after the
injections can be problematic especially where the amplitude
of the heat effect is close to background noise levels. The
analysis software supplied with ITC instruments can generally
do a reasonable job in this regard but may require “manual
adjustment” where there is drift, stepping, transient glitches, or
spurious points in the data. Other programs are available, such
as NITPIC, which have been developed to try and remove any
requirement for user input in this process [18]. Users are
encouraged to “experiment” with different manual and automated approaches. However, if the experimental result and
subsequent conclusions are in any way dependent on the way
in which the program or user has defined the baselines, then it
is clear that the experiment should be repeated and preferably
under conditions giving better signal amplitude that will eliminate any dependence on this element of the analysis process.
6. ITC instruments use a total cell fill mode, so that the cell
volume and protein concentration define how much material
is available. During the sequential injections of ligand, some of
the cell volume is displaced out into the filing tube and this is
prevented from re-equilibrating with cell material by the Teflon
plug on the syringe needle that sits in the neck of the cell. As a
consequence, the protein concentration in the cell will decrease
with each injection that increases the ligand concentration.
However, since the injection typically takes several seconds,
Isothermal Titration Calorimetry
153
immobilized on the sensor surface (irrespective of size) while
the other partner in the interaction is then termed the analyte.
3. If measurements are being made in solvent with a significantly
different heat capacity, e.g., buffers containing high salt concentration, organic solvents such as DMSO, or cryo preservatives such as glycerol, then the reference cell should be filled
with the same solvent.
4. Heat is being applied to both ITC cells during measurement
with a very small excess positive differential power being
applied to the sample cell. Since the cells are well insulated in
the ITC instrument, this energy input leads to a very small
increase in the absolute temperature of the cells over the duration of the measurement. However, this is very far from a level
significant for measurement accuracy and probably smaller
than temperature variations seen in many other techniques.
The temperature of measurement reported should be that set
in the instrument control software and used by the ITC during
its initial equilibration phase.
5. The integration of the excess peaks in differential power seen in
ITC data by defining baseline regions before and after the
injections can be problematic especially where the amplitude
of the heat effect is close to background noise levels. The
analysis software supplied with ITC instruments can generally
do a reasonable job in this regard but may require “manual
adjustment” where there is drift, stepping, transient glitches, or
spurious points in the data. Other programs are available, such
as NITPIC, which have been developed to try and remove any
requirement for user input in this process [18]. Users are
encouraged to “experiment” with different manual and automated approaches. However, if the experimental result and
subsequent conclusions are in any way dependent on the way
in which the program or user has defined the baselines, then it
is clear that the experiment should be repeated and preferably
under conditions giving better signal amplitude that will eliminate any dependence on this element of the analysis process.
6. ITC instruments use a total cell fill mode, so that the cell
volume and protein concentration define how much material
is available. During the sequential injections of ligand, some of
the cell volume is displaced out into the filing tube and this is
prevented from re-equilibrating with cell material by the Teflon
plug on the syringe needle that sits in the neck of the cell. As a
consequence, the protein concentration in the cell will decrease
with each injection that increases the ligand concentration.
However, since the injection typically takes several seconds,
Isothermal Titration Calorimetry
153