ITC cells is compatible with most buffers except strong acids.
The ITC instrument should be preferentially in a room with a
control temperature (about 20
C).
2. The buffer composition can influence the ITC signals. Reducing agents like DTT cause erratic baseline variations and TCEP
or β-mercaptoethanol are preferred to maintain reducing conditions, when needed. The ionization properties of the buffer
can have an influence on the values measured by ITC in particular when the interface involves charge exchange. In that case,
it is recommended to compare the values obtained with different buffers and at different ionic strengths (e.g., [23]).
3. If no heat exchange is observed during an interaction, it can
happen that the reaction exists but has a null enthalpy at the
temperature chosen. To evaluate this possibility, the ITC measurements can be performed at different temperatures (e.g.,
10
C lower or higher).
4. The ITC gives access to the stoichiometry (N) of the interaction. In many reactions, a value of 1 for N is expected, one
molecule of ligand bound by molecule of receptor. Even, in this
case, some deviation can be observed for several reasons:
(1) the presence of inactive receptors (unfolded, aggregated,
etc.) in the sample cell will lead to a lower N value, proportional
to the fraction of inactive receptor. The presence of inactive
ligands in the syringe will lead to a higher N value, proportional
to the fraction of inactive ligand. Similarly, errors on receptor
or ligand concentrations will have a direct impact on the
N value.
5. In parallel to the measurement of the receptor–ligand interaction, it is important to perform the ITC reaction with the
ligand in the syringe and buffer in the cell. This control will
evaluate if the signals observed are really due to the interaction
and not to other event like the dissociation of the ligand when
it is introduced in the sample cell. This can happen in particular
when the ligand is a protein that can form oligomers and
dissociate when injected in the cell.
6. Kinetic information can be recovered from ITC measurement
using a method called kinITC that can be applied for some
interactions
presenting
asymmetric
peaks
in
the
thermogram [24].
7. The range of K d accessible by ITC is classically between 0.1 nM
and 10 μM. For lower K d , the slope of the isotherm will be
sharp, preventing a precise measurement of the K d . However,
the ITC can be used in these conditions to determine ΔH and
N of the interaction. At higher K d , the measurement will often
need a large amount of receptors and ligands to have a correct
fit of the K d .
140
Amandine Gontier et al.
The ITC instrument should be preferentially in a room with a
control temperature (about 20
C).
2. The buffer composition can influence the ITC signals. Reducing agents like DTT cause erratic baseline variations and TCEP
or β-mercaptoethanol are preferred to maintain reducing conditions, when needed. The ionization properties of the buffer
can have an influence on the values measured by ITC in particular when the interface involves charge exchange. In that case,
it is recommended to compare the values obtained with different buffers and at different ionic strengths (e.g., [23]).
3. If no heat exchange is observed during an interaction, it can
happen that the reaction exists but has a null enthalpy at the
temperature chosen. To evaluate this possibility, the ITC measurements can be performed at different temperatures (e.g.,
10
C lower or higher).
4. The ITC gives access to the stoichiometry (N) of the interaction. In many reactions, a value of 1 for N is expected, one
molecule of ligand bound by molecule of receptor. Even, in this
case, some deviation can be observed for several reasons:
(1) the presence of inactive receptors (unfolded, aggregated,
etc.) in the sample cell will lead to a lower N value, proportional
to the fraction of inactive receptor. The presence of inactive
ligands in the syringe will lead to a higher N value, proportional
to the fraction of inactive ligand. Similarly, errors on receptor
or ligand concentrations will have a direct impact on the
N value.
5. In parallel to the measurement of the receptor–ligand interaction, it is important to perform the ITC reaction with the
ligand in the syringe and buffer in the cell. This control will
evaluate if the signals observed are really due to the interaction
and not to other event like the dissociation of the ligand when
it is introduced in the sample cell. This can happen in particular
when the ligand is a protein that can form oligomers and
dissociate when injected in the cell.
6. Kinetic information can be recovered from ITC measurement
using a method called kinITC that can be applied for some
interactions
presenting
asymmetric
peaks
in
the
thermogram [24].
7. The range of K d accessible by ITC is classically between 0.1 nM
and 10 μM. For lower K d , the slope of the isotherm will be
sharp, preventing a precise measurement of the K d . However,
the ITC can be used in these conditions to determine ΔH and
N of the interaction. At higher K d , the measurement will often
need a large amount of receptors and ligands to have a correct
fit of the K d .
140
Amandine Gontier et al.