then some of the protein that will be displaced can still participate in the binding and produce measured heat before it is
eventually ejected. The ITC instrument software accounts for
these complex effects and so it is best to use the concentrations
of protein and ligand that are tabulated in spreadsheet for
export of the data and fitting elsewhere.
7. It is common practice to include the small “pre-injection” in
ITC experiments eliminating premixed protein and ligand arising from diffusion during the equilibration phases of the
instrument as well as some unmixed ligand. In the iTC200,
this might be 0.5 μL while subsequent injections are 2 μL. The
concentration of ligand after this pre-injection is assumed to be
correct but the heat produced by the injection is reduced due
to some level of premixing and so it is not included as a data
point when fitting the titration. However, there is also the
possibility that the heat of this first injection is underestimated
due to mechanical backlash in the stepper motor that is used to
drive the syringe when it reverses direction following the loading process [19]. Solutions are drawn into the syringe from the
tip during loading and titrated back into the cell by driving the
system in the reverse direction. Thus, it is critical to make a
small movement in the direction of ligand ejection
(a “downward” movement in the iTC200 software) immediately after loading but before putting the syringe into the ITC
cell. Without this procedure, the pre-injection will take up the
backlash and give a smaller volume and incorrect ligand concentration after injection 1.
The use of approximately 20 injections during ITC titrations is generally engrained in the field as a “standard methodology” but there is evidence that the number of points in the
data set may be less important in constraining fitted parameters
than the amplitude of the heat measured for each injection. It
has been suggested that ten injections or even fewer may be a
more optimal experimental strategy, although despite some
considerable weight of literature in this area, this has not been
widely adopted [20] (and references therein). Advice would
generally be to initially use 20 injections to account for possible
complexity in the shape and features of the ITC binding data
but that for established systems with well characterized binding
behavior then a reduced number of larger injection volumes
should be considered.
An alternative approach to sequential injections and baseline recovery is to perform a single very slow injection of ligand
from the syringe [21]. The ITC signal is then contained in a
single differential power deflection that can be integrated and
analyzed to yield binding parameters. This single injection
methodology (SIM) has potential for some additional time
154
Christopher M. Johnson
eventually ejected. The ITC instrument software accounts for
these complex effects and so it is best to use the concentrations
of protein and ligand that are tabulated in spreadsheet for
export of the data and fitting elsewhere.
7. It is common practice to include the small “pre-injection” in
ITC experiments eliminating premixed protein and ligand arising from diffusion during the equilibration phases of the
instrument as well as some unmixed ligand. In the iTC200,
this might be 0.5 μL while subsequent injections are 2 μL. The
concentration of ligand after this pre-injection is assumed to be
correct but the heat produced by the injection is reduced due
to some level of premixing and so it is not included as a data
point when fitting the titration. However, there is also the
possibility that the heat of this first injection is underestimated
due to mechanical backlash in the stepper motor that is used to
drive the syringe when it reverses direction following the loading process [19]. Solutions are drawn into the syringe from the
tip during loading and titrated back into the cell by driving the
system in the reverse direction. Thus, it is critical to make a
small movement in the direction of ligand ejection
(a “downward” movement in the iTC200 software) immediately after loading but before putting the syringe into the ITC
cell. Without this procedure, the pre-injection will take up the
backlash and give a smaller volume and incorrect ligand concentration after injection 1.
The use of approximately 20 injections during ITC titrations is generally engrained in the field as a “standard methodology” but there is evidence that the number of points in the
data set may be less important in constraining fitted parameters
than the amplitude of the heat measured for each injection. It
has been suggested that ten injections or even fewer may be a
more optimal experimental strategy, although despite some
considerable weight of literature in this area, this has not been
widely adopted [20] (and references therein). Advice would
generally be to initially use 20 injections to account for possible
complexity in the shape and features of the ITC binding data
but that for established systems with well characterized binding
behavior then a reduced number of larger injection volumes
should be considered.
An alternative approach to sequential injections and baseline recovery is to perform a single very slow injection of ligand
from the syringe [21]. The ITC signal is then contained in a
single differential power deflection that can be integrated and
analyzed to yield binding parameters. This single injection
methodology (SIM) has potential for some additional time
154
Christopher M. Johnson