It is possible using a more complicated set of equations to
account for the effect of the difference in fluorescence intensity of
free and bound HEWL-D488 on the signal averaging in the thermophoresis experiments (see Note 27). These equations were used
to globally fit the five MST datasets, accounting for the mean
fluorescence intensity increase of 1.6-fold observed upon binding
of NAG3 to HEWL-D488. This gave a shared K d value of 10.0 μM
with a 95% confidence interval of 8.9–11.2 μM.
It was not possible to use this system of equations to fit both
the fluorescence intensity data and the MST data for NAG3 binding to HEWL-D488 to a common model with a shared value of K d .
The K d measured by fluorescence intensity is apparently significantly higher than that measured by MST. It is not clear at present
why this is the case. It is probable either (1) that there are additional
binding events, or changes in solution properties, that take place at
high NAG3 concentrations and are not related to the binding event
that is monitored by MST; or (2) that there is some heterogeneity
in the location of the dye within the population of HEWL-D488,
giving rise to different fluorescence intensity changes and different
K d values for different subpopulations (see Note 28).
3.5 Isothermal
Titration
Calorimetry (ITC)
3.5.1 ITC Measurement
The aim is to perform an ITC titration in the standard configuration for small-molecule binding, titrating NAG3 (in the injector
syringe) against HEWL (in the calorimeter cell). Essentially the
same experiment is discussed in detail, together with much useful
advice and many recommendations for best practice, in Chapter 5.
1. Consider performing a thorough cleaning of the instrument
with suitable detergent solution at elevated temperature, followed by a water vs. water titration to confirm that the instrument is functional and that the level of noise arising from
contamination or mechanical imperfection is low.
2. Prepare an appropriate volume of solution of 35 μM HEWL
and a solution of 400 μM NAG3 (see Note 29) in standard
assay buffer, in a volume suitable for the instrument that you
are using (see Note 30).
3. Set the temperature of the calorimeter to 25
C.
4. For the larger cell-volume instruments, degas both the HEWL
and NAG3 solutions for 5 min under a weak vacuum. For the
smaller cell-volume instrument, it is usually sufficient only to
degas the cell solution. If possible, degassing should be performed slightly below the desired experimental temperature.
5. Wash the cell-loading syringe, the calorimeter cell, and the
injector syringe extensively with filtered distilled water, and
check that the reference cell is filled with degassed distilled
water.
64
Xiaochun Li-Blatter et al.
account for the effect of the difference in fluorescence intensity of
free and bound HEWL-D488 on the signal averaging in the thermophoresis experiments (see Note 27). These equations were used
to globally fit the five MST datasets, accounting for the mean
fluorescence intensity increase of 1.6-fold observed upon binding
of NAG3 to HEWL-D488. This gave a shared K d value of 10.0 μM
with a 95% confidence interval of 8.9–11.2 μM.
It was not possible to use this system of equations to fit both
the fluorescence intensity data and the MST data for NAG3 binding to HEWL-D488 to a common model with a shared value of K d .
The K d measured by fluorescence intensity is apparently significantly higher than that measured by MST. It is not clear at present
why this is the case. It is probable either (1) that there are additional
binding events, or changes in solution properties, that take place at
high NAG3 concentrations and are not related to the binding event
that is monitored by MST; or (2) that there is some heterogeneity
in the location of the dye within the population of HEWL-D488,
giving rise to different fluorescence intensity changes and different
K d values for different subpopulations (see Note 28).
3.5 Isothermal
Titration
Calorimetry (ITC)
3.5.1 ITC Measurement
The aim is to perform an ITC titration in the standard configuration for small-molecule binding, titrating NAG3 (in the injector
syringe) against HEWL (in the calorimeter cell). Essentially the
same experiment is discussed in detail, together with much useful
advice and many recommendations for best practice, in Chapter 5.
1. Consider performing a thorough cleaning of the instrument
with suitable detergent solution at elevated temperature, followed by a water vs. water titration to confirm that the instrument is functional and that the level of noise arising from
contamination or mechanical imperfection is low.
2. Prepare an appropriate volume of solution of 35 μM HEWL
and a solution of 400 μM NAG3 (see Note 29) in standard
assay buffer, in a volume suitable for the instrument that you
are using (see Note 30).
3. Set the temperature of the calorimeter to 25
C.
4. For the larger cell-volume instruments, degas both the HEWL
and NAG3 solutions for 5 min under a weak vacuum. For the
smaller cell-volume instrument, it is usually sufficient only to
degas the cell solution. If possible, degassing should be performed slightly below the desired experimental temperature.
5. Wash the cell-loading syringe, the calorimeter cell, and the
injector syringe extensively with filtered distilled water, and
check that the reference cell is filled with degassed distilled
water.
64
Xiaochun Li-Blatter et al.