4. Analyze samples with a Safire
2
™ plate reader and adjust instrumental z-factor at maximum signal. Calibrate g-factor with
BSA blanks and buffer controls to target an initial millipolarization (mP) of 20 mP.
5. Record measurements at excitation/emission wavelengths of
λ 633 nm /λ 647 nm at 25
C. After subtracting background of
20 mP, fit saturation data using one-site specific binding
model with GraphPad Prism mathematical software. Determine K d by identifying protein concentration at halfsaturation.
3.3.2 Isothermal
Calorimetry (ITC)
ITC is another in vitro assay that provides in addition to the
binding affinity, thermodynamic data on the binding mechanism
that can be useful in ligand development. Moreover, this technique
is label free in comparison to FP where a fluorophore needs to be
conjugated to the ligand.
1. The ligand and the protein must be solubilized in the same
buffer PBS and pH adjusted to 7.4 within 0.02 pH units (see
Note 15).
2. The calorimetry experiments were performed at 25
C, by
titration of the ligand (20 Â 2 μL injections at 180 s intervals;
stirring speed of 1000 rpm) into the PDZ solution.
3. The Experiments were designed so that c-values (c-value ¼ K a *
[Protein]*N; where K a is the affinity association constant,
[Protein] is the protein concentration, and N is the stoichiometry of the binding event) were generally within 1–1000.
4. Heats of dilution were determined by titrating the ligand into
buffer and buffer into protein, respectively, which were subtracted from the observed “heat values” of ligand into protein.
5. Use ORIGIN 7.0 (Malvern Pananalytical Ltd., Malvern, UK)
to determined the thermodynamic properties of ligand binding
using nonlinear least-squares fitting assuming a single-site
model.
3.4 Further
Validation
Experiments
3.4.1 Pull-Down
of Nonischemic Brain
Lysates
Pull-down assays are used to probe protein–protein interactions in a
more complex environment. The experiment is based on a bait
molecule that is immobilized on a solid support, such as dextran
or agarose, and a mixture of potential interaction partners, such as a
tissue lysate. After incubation of bait and pray, the nonbinding
molecules are washed, and the binding molecules are eluted and
further analyzed.
1. Synthesis and purification of biotin-labeled PSD-95 inhibitor
as described in Subheading 3.1.1 solid phase peptide synthesis
(see Note 16).
170
Dominik J. Essig et al.
2
™ plate reader and adjust instrumental z-factor at maximum signal. Calibrate g-factor with
BSA blanks and buffer controls to target an initial millipolarization (mP) of 20 mP.
5. Record measurements at excitation/emission wavelengths of
λ 633 nm /λ 647 nm at 25
C. After subtracting background of
20 mP, fit saturation data using one-site specific binding
model with GraphPad Prism mathematical software. Determine K d by identifying protein concentration at halfsaturation.
3.3.2 Isothermal
Calorimetry (ITC)
ITC is another in vitro assay that provides in addition to the
binding affinity, thermodynamic data on the binding mechanism
that can be useful in ligand development. Moreover, this technique
is label free in comparison to FP where a fluorophore needs to be
conjugated to the ligand.
1. The ligand and the protein must be solubilized in the same
buffer PBS and pH adjusted to 7.4 within 0.02 pH units (see
Note 15).
2. The calorimetry experiments were performed at 25
C, by
titration of the ligand (20 Â 2 μL injections at 180 s intervals;
stirring speed of 1000 rpm) into the PDZ solution.
3. The Experiments were designed so that c-values (c-value ¼ K a *
[Protein]*N; where K a is the affinity association constant,
[Protein] is the protein concentration, and N is the stoichiometry of the binding event) were generally within 1–1000.
4. Heats of dilution were determined by titrating the ligand into
buffer and buffer into protein, respectively, which were subtracted from the observed “heat values” of ligand into protein.
5. Use ORIGIN 7.0 (Malvern Pananalytical Ltd., Malvern, UK)
to determined the thermodynamic properties of ligand binding
using nonlinear least-squares fitting assuming a single-site
model.
3.4 Further
Validation
Experiments
3.4.1 Pull-Down
of Nonischemic Brain
Lysates
Pull-down assays are used to probe protein–protein interactions in a
more complex environment. The experiment is based on a bait
molecule that is immobilized on a solid support, such as dextran
or agarose, and a mixture of potential interaction partners, such as a
tissue lysate. After incubation of bait and pray, the nonbinding
molecules are washed, and the binding molecules are eluted and
further analyzed.
1. Synthesis and purification of biotin-labeled PSD-95 inhibitor
as described in Subheading 3.1.1 solid phase peptide synthesis
(see Note 16).
170
Dominik J. Essig et al.