instrument over time), and SPR data (where the saturating biosensor response is proportional to the amount of HEWL immobilized,
which varies between experiments). However, for ITC instruments
from a single manufacturer, it might be expected that the calibration and measurement of differential power are sufficiently accurate
and precise that the enthalpies of binding should be consistent
between instruments. Therefore, in globally fitting the ITC data
presented here, both the K d and ΔH are fitted as global parameters.
Further details of the fitting are provided in later sections.
On advantage of fitting K d as a global parameter for all five
datasets for each technique is that it allows the method of error
surface projection (ESP) to be used to calculate a single confidence
interval for that global K d , taking into account all of the variability
in the replicates. There is insufficient space to cover this method in
detail here, but it has been widely employed for determination of
confidence limits on fitted parameters and is described in more
detail elsewhere [20]. Essentially it calculates the range of one fitted
parameter that can fit the data at a given confidence limit, if other
floating parameters are allowed to vary. The confidence limits
derived in this way can be asymmetric about the best-fit value, in
contrast to other methods that assume a Gaussian error distribution. This approach of global fitting and error analysis using ESP
seems the most straightforward way to include many sources of
experimental error into a realistic estimate of the confidence in a
single parameter of interest measured by a single technique.
2 Materials
2.1 Buffers
l
Standard assay buffer: 0.1 M sodium acetate/acetic acid, pH 5.0
(see Note 4).
l
MST Buffer: 0.1 M sodium acetate/acetic acid, pH 5.0, 0.1%
v/v Tween 20 (see Note 5).
l
MST labeling buffer: 50 mM sodium phosphate, pH 7.5,
150 mM NaCl.
l
SPR immobilization buffer: 50 mM sodium phosphate, pH 8.5.
2.2 Protein
and Ligand
l
Hen egg white lysozyme (HEWL): Molar mass: 14,313 g/mol,
ε 280 37,970 M
À1 cm
À1 .
l
Tri-N-acetyl glucosamine (NAG3): Molar mass: 627.6 g/mol
(see Note 6).
l
Various stock concentrations of HEWL and NAG3 are used in
this chapter to suit different types of experiment. For detailed
preparation instructions, please see Subheading 3.1.
52
Xiaochun Li-Blatter et al.
which varies between experiments). However, for ITC instruments
from a single manufacturer, it might be expected that the calibration and measurement of differential power are sufficiently accurate
and precise that the enthalpies of binding should be consistent
between instruments. Therefore, in globally fitting the ITC data
presented here, both the K d and ΔH are fitted as global parameters.
Further details of the fitting are provided in later sections.
On advantage of fitting K d as a global parameter for all five
datasets for each technique is that it allows the method of error
surface projection (ESP) to be used to calculate a single confidence
interval for that global K d , taking into account all of the variability
in the replicates. There is insufficient space to cover this method in
detail here, but it has been widely employed for determination of
confidence limits on fitted parameters and is described in more
detail elsewhere [20]. Essentially it calculates the range of one fitted
parameter that can fit the data at a given confidence limit, if other
floating parameters are allowed to vary. The confidence limits
derived in this way can be asymmetric about the best-fit value, in
contrast to other methods that assume a Gaussian error distribution. This approach of global fitting and error analysis using ESP
seems the most straightforward way to include many sources of
experimental error into a realistic estimate of the confidence in a
single parameter of interest measured by a single technique.
2 Materials
2.1 Buffers
l
Standard assay buffer: 0.1 M sodium acetate/acetic acid, pH 5.0
(see Note 4).
l
MST Buffer: 0.1 M sodium acetate/acetic acid, pH 5.0, 0.1%
v/v Tween 20 (see Note 5).
l
MST labeling buffer: 50 mM sodium phosphate, pH 7.5,
150 mM NaCl.
l
SPR immobilization buffer: 50 mM sodium phosphate, pH 8.5.
2.2 Protein
and Ligand
l
Hen egg white lysozyme (HEWL): Molar mass: 14,313 g/mol,
ε 280 37,970 M
À1 cm
À1 .
l
Tri-N-acetyl glucosamine (NAG3): Molar mass: 627.6 g/mol
(see Note 6).
l
Various stock concentrations of HEWL and NAG3 are used in
this chapter to suit different types of experiment. For detailed
preparation instructions, please see Subheading 3.1.
52
Xiaochun Li-Blatter et al.