4 Notes
1. The conditions for the ITC experiment were initially found in
lecture notes from Professor Alan Cooper and were used as a
starting point for all other experiments. Calorimetric studies of
the interaction have also been reported in the literature under
these conditions [7] and under slightly different
conditions [6].
2. It is important that only the binding partner present at a
constant concentration in a titration exhibits fluorescence in
the wavelength range used. Depending on the type of instrument used and the experimental context, either intrinsic protein fluorescence or fluorescence from an extrinsic dye can be
used. The choice to use an extrinsic dye in the case presented in
this chapter was determined solely by the capabilites of the
available instrument.
3. The data were collected under supervision from expert users,
and the graduate students who participated in the course were
also engaged in collection of data for publication. Therfore, it
might be argued that the data offer a realistic representation of
nonexpert use after training.
4. pH 5 acetate/acetic acid is used for three reasons: firstly, it is
the pH at which the affinity of the interaction is reported to be
highest; secondly, it is below the pH at which significant populations of dimer form for lysozyme [24]; and thirdly, the
enthalpy of ionization of acetate/acetic acid buffers is essentially zero, meaning that they do not change pH significantly
with temperature (useful for the thermal shift assay) and do not
contribute to observed enthalpies of binding if proton-transfer
occurs. All the buffers should ideally be prepared by weighing
appropriate amounts of acidic and basic buffer species and
dissolving them in an appropriate volume of pure distilled
water, calculated taking into account the intended working
temperature. An excellent resource for such calculations is
www.liverpool.ac.uk/pfg/Research/Tools/BuffferCalc/
Buffer.html. For example, to make 1 L of the standard assay
buffer for use at 25
C, 0.0314 mol acetic acid and 0.0685 mol
sodium acetate should be dissolved in a total 1 L solution in a
volumetric flask. It is also possible to prepare the buffer by
carefully titrating a solution of 0.1 M sodium acetate to pH
5.0 using concentrated HCl, monitored by a calibrated pH
meter. The buffer should be filtered through a 0.22-μm filter
to sterilize it and remove particulate matter before use in
experiments.
5. The MST experiment uses a low nanomolar concentration of
fluorescently labeled lysozyme solution. To suppress binding of
protein to plastic and glass surfaces during sample preparation
Interactions by Multiple Methods
71
1. The conditions for the ITC experiment were initially found in
lecture notes from Professor Alan Cooper and were used as a
starting point for all other experiments. Calorimetric studies of
the interaction have also been reported in the literature under
these conditions [7] and under slightly different
conditions [6].
2. It is important that only the binding partner present at a
constant concentration in a titration exhibits fluorescence in
the wavelength range used. Depending on the type of instrument used and the experimental context, either intrinsic protein fluorescence or fluorescence from an extrinsic dye can be
used. The choice to use an extrinsic dye in the case presented in
this chapter was determined solely by the capabilites of the
available instrument.
3. The data were collected under supervision from expert users,
and the graduate students who participated in the course were
also engaged in collection of data for publication. Therfore, it
might be argued that the data offer a realistic representation of
nonexpert use after training.
4. pH 5 acetate/acetic acid is used for three reasons: firstly, it is
the pH at which the affinity of the interaction is reported to be
highest; secondly, it is below the pH at which significant populations of dimer form for lysozyme [24]; and thirdly, the
enthalpy of ionization of acetate/acetic acid buffers is essentially zero, meaning that they do not change pH significantly
with temperature (useful for the thermal shift assay) and do not
contribute to observed enthalpies of binding if proton-transfer
occurs. All the buffers should ideally be prepared by weighing
appropriate amounts of acidic and basic buffer species and
dissolving them in an appropriate volume of pure distilled
water, calculated taking into account the intended working
temperature. An excellent resource for such calculations is
www.liverpool.ac.uk/pfg/Research/Tools/BuffferCalc/
Buffer.html. For example, to make 1 L of the standard assay
buffer for use at 25
C, 0.0314 mol acetic acid and 0.0685 mol
sodium acetate should be dissolved in a total 1 L solution in a
volumetric flask. It is also possible to prepare the buffer by
carefully titrating a solution of 0.1 M sodium acetate to pH
5.0 using concentrated HCl, monitored by a calibrated pH
meter. The buffer should be filtered through a 0.22-μm filter
to sterilize it and remove particulate matter before use in
experiments.
5. The MST experiment uses a low nanomolar concentration of
fluorescently labeled lysozyme solution. To suppress binding of
protein to plastic and glass surfaces during sample preparation
Interactions by Multiple Methods
71