established technique in the lab but there is no recognized expert
available to give instruction, then the instrument manual and the
instructions below are a good starting point.
This chapter attempts to establish good basic experimental
practice and then to build on this initial platform to hopefully
confer additional experience gained over the many years of ITC
use in many different biological settings. It considers the real
practical aspects of ITC that are encountered when working with
proteins and details test chemistry that uses a protein–ligand interaction that has been verified using other techniques that might be
available for comparison (see Chapter 2). The numerous advantages
and unique insights given by ITC are considered but not forgetting
also to discuss problem areas and practical limitations of the technique. Data processing, fitting issues, and data accuracy are also
covered so that the results obtained from ITC can be set in a reliable
framework and used in a realistic way.
2 Materials
2.1 ITC Instrumental
Basics
The MicroCal iTC200 instrument has two metal cells, sample and
reference, that are located in the core of the instrument in an
insulated adiabatic environment (see Note 1). The cells cannot be
seen or removed and are accessed through filling tubes that connect
to the surface of the instrument as illustrated in Fig. 1.
The sample cell has a syringe titration system that can be
introduced via the filling tube. The syringe has a paddle at its end
(either twisted, as shown in Fig. 1, or flat) and is rotated at 750 or
1000 rpm to mix the cell contents very efficiently. It can make
small μL volume injections of ligand using a stepper motor that
drives the plunger (see Note 2). The reference cell is typically filled
with water (see Note 3).
The instrument maintains a small difference in temperature
between these two cells using a number of electrical heaters
attached to their surface. The heaters are driven in a feedback
loop from the voltage output of a very precise thermopile that is
measuring the temperature difference as it is arranged between the
two cells. It is then levels of excess differential power applied to the
sample cell that become the instrument output varying during a
titration as ligand is injected, binding occurs, and heat is produced
or absorbed. By maintaining a positive level of differential power to
the sample cell during measurement, the instrument can increase
further this energy input, in the case of heat-absorbing endothermic (ΔH +ve) events on addition of ligand by the syringe, or it can
reduce the energy input in the event of heat-producing exothermic
(ΔH Àve) events (see Note 4). The instrument thus has an upper
limit of measurement, where the differential power circuit is
Isothermal Titration Calorimetry
137
available to give instruction, then the instrument manual and the
instructions below are a good starting point.
This chapter attempts to establish good basic experimental
practice and then to build on this initial platform to hopefully
confer additional experience gained over the many years of ITC
use in many different biological settings. It considers the real
practical aspects of ITC that are encountered when working with
proteins and details test chemistry that uses a protein–ligand interaction that has been verified using other techniques that might be
available for comparison (see Chapter 2). The numerous advantages
and unique insights given by ITC are considered but not forgetting
also to discuss problem areas and practical limitations of the technique. Data processing, fitting issues, and data accuracy are also
covered so that the results obtained from ITC can be set in a reliable
framework and used in a realistic way.
2 Materials
2.1 ITC Instrumental
Basics
The MicroCal iTC200 instrument has two metal cells, sample and
reference, that are located in the core of the instrument in an
insulated adiabatic environment (see Note 1). The cells cannot be
seen or removed and are accessed through filling tubes that connect
to the surface of the instrument as illustrated in Fig. 1.
The sample cell has a syringe titration system that can be
introduced via the filling tube. The syringe has a paddle at its end
(either twisted, as shown in Fig. 1, or flat) and is rotated at 750 or
1000 rpm to mix the cell contents very efficiently. It can make
small μL volume injections of ligand using a stepper motor that
drives the plunger (see Note 2). The reference cell is typically filled
with water (see Note 3).
The instrument maintains a small difference in temperature
between these two cells using a number of electrical heaters
attached to their surface. The heaters are driven in a feedback
loop from the voltage output of a very precise thermopile that is
measuring the temperature difference as it is arranged between the
two cells. It is then levels of excess differential power applied to the
sample cell that become the instrument output varying during a
titration as ligand is injected, binding occurs, and heat is produced
or absorbed. By maintaining a positive level of differential power to
the sample cell during measurement, the instrument can increase
further this energy input, in the case of heat-absorbing endothermic (ΔH +ve) events on addition of ligand by the syringe, or it can
reduce the energy input in the event of heat-producing exothermic
(ΔH Àve) events (see Note 4). The instrument thus has an upper
limit of measurement, where the differential power circuit is
Isothermal Titration Calorimetry
137