3 Methods
To get an accurate K d measurement, the receptor and the ligand
concentrations should be known accurately. This can be determined with the UV spectrum of both protein and DNA samples
and their extinction coefficient. The biophysical measurement of
the K d by ITC or MST (as with many other biophysical approaches)
requires highly purified samples. This implies that quality control
(QC) analyses are performed before measurements and that the QC
indicates high purity (greater than 95%). Protein purity can be
determined using several methods including UV spectrum, SDS
PAGE gels, and analytical chromatography (size exclusion chromatography or anion exchange) [21]. DNA purity can be analyzed by
using UV spectrum, acrylamide gels, or analytical anion exchange
chromatography. The receptor is commonly used at a fixed concentration, and the ligand is used at various concentrations ranging
from 1/10 to 20-fold the expected K d . The way to define the
concentrations used for the receptor in ITC and MST is described
below according to the specificities of these two methods.
3.1 Methods for ITC
To get an accurate K d measurement with ITC, the receptor concentration in the cell of the instrument and the ligand loaded in the
syringe should be well chosen. The receptor concentration appropriate for an ITC experiment can be estimated using the dimensionless constant c, determined by the formula, c ¼ N  K a  [Ro],
where N is the stoichiometry of the reaction (number of ligand
molecules bound by molecule of receptor), K a is the association
constant (M
À1
), and [Ro] the initial concentration of the receptor
in the sample cell (M). To obtain a well-defined S-shaped binding
isotherm with two plateau (Fig. 1b, bottom) and an accurate
measurement of the thermodynamic parameters of the equilibrium,
it is recommended to use an initial concentration of the receptor
[Ro] that results in a c value between 20 and 200 [22]. In the
absence of a priori information, typical starting concentrations are
1 μM for the receptor and 10 μM for the ligand. The ligand is
usually loaded in the syringe at a concentration ten times higher
than the receptor, so that at the end of the titration (typically
28 injections of 10 μl), the molar ratio of the ligand over the
receptor is 2.
3.1.1 Experimental
Design
In our example Ku–DNA, with an expected K d of about 5 nM and a
stoichiometry N of 1, we started with a concentration of Ku in the
cell ([Ro]) of 1 μM corresponding to a c value of 200. The ligand
and the receptor are dialyzed against the same buffer to minimize
heat exchanges due to buffer differences.
Measurements of Protein–DNA Complexes Interactions by Isothermal. . .
133
To get an accurate K d measurement, the receptor and the ligand
concentrations should be known accurately. This can be determined with the UV spectrum of both protein and DNA samples
and their extinction coefficient. The biophysical measurement of
the K d by ITC or MST (as with many other biophysical approaches)
requires highly purified samples. This implies that quality control
(QC) analyses are performed before measurements and that the QC
indicates high purity (greater than 95%). Protein purity can be
determined using several methods including UV spectrum, SDS
PAGE gels, and analytical chromatography (size exclusion chromatography or anion exchange) [21]. DNA purity can be analyzed by
using UV spectrum, acrylamide gels, or analytical anion exchange
chromatography. The receptor is commonly used at a fixed concentration, and the ligand is used at various concentrations ranging
from 1/10 to 20-fold the expected K d . The way to define the
concentrations used for the receptor in ITC and MST is described
below according to the specificities of these two methods.
3.1 Methods for ITC
To get an accurate K d measurement with ITC, the receptor concentration in the cell of the instrument and the ligand loaded in the
syringe should be well chosen. The receptor concentration appropriate for an ITC experiment can be estimated using the dimensionless constant c, determined by the formula, c ¼ N  K a  [Ro],
where N is the stoichiometry of the reaction (number of ligand
molecules bound by molecule of receptor), K a is the association
constant (M
À1
), and [Ro] the initial concentration of the receptor
in the sample cell (M). To obtain a well-defined S-shaped binding
isotherm with two plateau (Fig. 1b, bottom) and an accurate
measurement of the thermodynamic parameters of the equilibrium,
it is recommended to use an initial concentration of the receptor
[Ro] that results in a c value between 20 and 200 [22]. In the
absence of a priori information, typical starting concentrations are
1 μM for the receptor and 10 μM for the ligand. The ligand is
usually loaded in the syringe at a concentration ten times higher
than the receptor, so that at the end of the titration (typically
28 injections of 10 μl), the molar ratio of the ligand over the
receptor is 2.
3.1.1 Experimental
Design
In our example Ku–DNA, with an expected K d of about 5 nM and a
stoichiometry N of 1, we started with a concentration of Ku in the
cell ([Ro]) of 1 μM corresponding to a c value of 200. The ligand
and the receptor are dialyzed against the same buffer to minimize
heat exchanges due to buffer differences.
Measurements of Protein–DNA Complexes Interactions by Isothermal. . .
133