requires very small amounts of material—typically in the 100 ng to
1 μg range; (2) there is almost no limitation to molecular size or
mass; (3) the method is easy and fast to perform; and (4) it is an
immobilization free method. MST has also several limitations:
(1) one of the partners should be labeled with possible steric
hindrance with the interaction sites, and hydrophobic fluorescent
probes may cause some nonspecific binding; and (2) the theory
behind the measurement principle is less obvious than for other
biophysical approaches.
2 Materials
2.1 Equipment
for Isothermal Titration
Calorimetry (ITC)
1. MicroCal VP-ITC: The VP-ITC microcalorimeter (Malvern) is
composed of two compartments or cells: one reference cell
is filled with water or buffer and one measurement cell (volume
of 1.4 ml) is filled with the first partner called the receptor.
Both are maintained at equal temperature and are positioned in
an adiabatic environment. The ligand is introduced with small
injections (5–10 μl) in the measurement cell. If the ligand
interacts with the receptor, in most cases, some heat is generated or absorbed due to the interaction. This is detected by the
device that compensates by respectively reducing or increasing
the electric current in the measurement cell (Fig. 1). In some
rare cases, at a given temperature, an interaction can happen
without heat exchange (ΔH ¼ 0). In that case, changing the
temperature (typically Æ10
C) should unmask the interaction,
if it exists, since the enthalpy varies with the temperature.
2. The ITC-200 microcalorimeter which is equipped with a measurement cell of 200 μl and a syringe of 40 μl requires smaller
quantities of sample. In the case of the Ku–DNA interaction,
presented here, the VP-ITC calorimeter provided better thermograms and a better signal/noise ratio.
3. ThermoVac™ (Malvern): This device enables to degas the
buffers and samples before use and to pre-equilibrate at a
temperature close to the measurement temperature. It is also
used as pump for cleaning procedures.
4. VPViewer™ and Origin™ software packages: VPViewer is
used to command the VP-ITC and to follow the experiment.
Origin is used to analyze the raw data called thermograms.
2.2 Samples for ITC
As will be detailed below, suitable concentrations for a titration
depend on the expected K d between the ligand and the receptor
and on the enthalpy associated with the formation of the complex.
Quantities listed below are required to characterize the interaction
between the Ku70–Ku80 (Ku) heterodimer and small DNA
130
Amandine Gontier et al.
1 μg range; (2) there is almost no limitation to molecular size or
mass; (3) the method is easy and fast to perform; and (4) it is an
immobilization free method. MST has also several limitations:
(1) one of the partners should be labeled with possible steric
hindrance with the interaction sites, and hydrophobic fluorescent
probes may cause some nonspecific binding; and (2) the theory
behind the measurement principle is less obvious than for other
biophysical approaches.
2 Materials
2.1 Equipment
for Isothermal Titration
Calorimetry (ITC)
1. MicroCal VP-ITC: The VP-ITC microcalorimeter (Malvern) is
composed of two compartments or cells: one reference cell
is filled with water or buffer and one measurement cell (volume
of 1.4 ml) is filled with the first partner called the receptor.
Both are maintained at equal temperature and are positioned in
an adiabatic environment. The ligand is introduced with small
injections (5–10 μl) in the measurement cell. If the ligand
interacts with the receptor, in most cases, some heat is generated or absorbed due to the interaction. This is detected by the
device that compensates by respectively reducing or increasing
the electric current in the measurement cell (Fig. 1). In some
rare cases, at a given temperature, an interaction can happen
without heat exchange (ΔH ¼ 0). In that case, changing the
temperature (typically Æ10
C) should unmask the interaction,
if it exists, since the enthalpy varies with the temperature.
2. The ITC-200 microcalorimeter which is equipped with a measurement cell of 200 μl and a syringe of 40 μl requires smaller
quantities of sample. In the case of the Ku–DNA interaction,
presented here, the VP-ITC calorimeter provided better thermograms and a better signal/noise ratio.
3. ThermoVac™ (Malvern): This device enables to degas the
buffers and samples before use and to pre-equilibrate at a
temperature close to the measurement temperature. It is also
used as pump for cleaning procedures.
4. VPViewer™ and Origin™ software packages: VPViewer is
used to command the VP-ITC and to follow the experiment.
Origin is used to analyze the raw data called thermograms.
2.2 Samples for ITC
As will be detailed below, suitable concentrations for a titration
depend on the expected K d between the ligand and the receptor
and on the enthalpy associated with the formation of the complex.
Quantities listed below are required to characterize the interaction
between the Ku70–Ku80 (Ku) heterodimer and small DNA
130
Amandine Gontier et al.