techniques [16, 17]. However, the thermodynamics of binding are
very hard to interpret unambiguously from a single experiment, so
in this set of experiments, the primary use of ITC is to measure the
dissociation constant for the interaction. For further details about
ITC, readers should consult Chapter 5.
Surface plasmon resonance (SPR): This technique measures the
properties of light reflected from a metal–aqueous interface, which
are sensitive to the refractive index in the aqueous layer at the metal
surface [18, 19]. If one binding partner (the “ligand” in SPR
terminology) is immobilized at the surface and another binding
partner (the “analyte”) is introduced in flow across the surface, the
refractive index in the aqueous layer changes as the analyte is
accumulated due to interaction with the ligand, giving rise to a
measurable change in the properties of the reflected light. By alternating between flow containing analyte, and flow without analyte,
an association phase, an equilibrium response, and a dissociation
phase can be observed. For interactions with association and dissociation rates in the right range, information on both binding kinetics and dissociation constants can be obtained. However, for the
interaction presented here, the kinetics are too fast to be measured
by the instrument at concentrations that give measurable signal
changes, and so the rate constants cannot be obtained. Therefore,
the focus of the experiment is to obtain the dissociation constant
for the interaction from the steady-state value of the instrumental
response. For further details about SPR, readers may consult
Chapter 17.
For the sake of brevity, only those technical details necessary to
ensure successful replication of the experiments will be included,
together with typical results taken in the most part from student
practicals, perhaps the ultimate testing ground for experimental
robustness and replicability. Some of the experimental details are
necessarily instrument-specific, and it is hoped that readers can use
these as a starting point for adaptation to their own instruments.
The experimental protocols should not be regarded as optimized, nor should the results be regarded as definitive, though
most of the protocols have been successfully reproduced in different labs. Rather, it is hoped that this information will prove helpful
to others engaged in practical teaching of biophysics or embarking
on a project to quantify a protein–ligand interaction, and that this
chapter might serve as a starting point for further optimization and
expansion.
1.4 Nature
and Analysis
of Example Data
To illustrate the experimental protocols and to give an indication of
their robustness, this chapter will present and analyze five or more
datasets for each technique, in most cases collected during student
practical classes in the last 5 years. As might be expected for student
practicals, the standards of pipetting and sample-handling were
somewhat variable. Datasets were selected essentially at random,
50
Xiaochun Li-Blatter et al.
very hard to interpret unambiguously from a single experiment, so
in this set of experiments, the primary use of ITC is to measure the
dissociation constant for the interaction. For further details about
ITC, readers should consult Chapter 5.
Surface plasmon resonance (SPR): This technique measures the
properties of light reflected from a metal–aqueous interface, which
are sensitive to the refractive index in the aqueous layer at the metal
surface [18, 19]. If one binding partner (the “ligand” in SPR
terminology) is immobilized at the surface and another binding
partner (the “analyte”) is introduced in flow across the surface, the
refractive index in the aqueous layer changes as the analyte is
accumulated due to interaction with the ligand, giving rise to a
measurable change in the properties of the reflected light. By alternating between flow containing analyte, and flow without analyte,
an association phase, an equilibrium response, and a dissociation
phase can be observed. For interactions with association and dissociation rates in the right range, information on both binding kinetics and dissociation constants can be obtained. However, for the
interaction presented here, the kinetics are too fast to be measured
by the instrument at concentrations that give measurable signal
changes, and so the rate constants cannot be obtained. Therefore,
the focus of the experiment is to obtain the dissociation constant
for the interaction from the steady-state value of the instrumental
response. For further details about SPR, readers may consult
Chapter 17.
For the sake of brevity, only those technical details necessary to
ensure successful replication of the experiments will be included,
together with typical results taken in the most part from student
practicals, perhaps the ultimate testing ground for experimental
robustness and replicability. Some of the experimental details are
necessarily instrument-specific, and it is hoped that readers can use
these as a starting point for adaptation to their own instruments.
The experimental protocols should not be regarded as optimized, nor should the results be regarded as definitive, though
most of the protocols have been successfully reproduced in different labs. Rather, it is hoped that this information will prove helpful
to others engaged in practical teaching of biophysics or embarking
on a project to quantify a protein–ligand interaction, and that this
chapter might serve as a starting point for further optimization and
expansion.
1.4 Nature
and Analysis
of Example Data
To illustrate the experimental protocols and to give an indication of
their robustness, this chapter will present and analyze five or more
datasets for each technique, in most cases collected during student
practical classes in the last 5 years. As might be expected for student
practicals, the standards of pipetting and sample-handling were
somewhat variable. Datasets were selected essentially at random,
50
Xiaochun Li-Blatter et al.