5. Gently mix the sample using a pipette being careful to avoid
introducing bubbles (see Note 9).
6. Cover the cuvette to prevent introducing dust particles and to
minimize the dansyl-fluorophore light exposure (see Note 10).
3.2.2 Preparation of PDZ
Domain Dilution Stock
Solutions
All the PDZ domain diluted solutions described below should be
prepared in advance and on ice.
1. Prepare 26 μL of a 100-fold diluted PDZ domain stock per
experiment (concentration 5–20 μM).
2. Prepare 10 μL of a tenfold diluted PDZ domain stock per
experiment (concentration 50–200 μM).
3. Prepare 656 μL PDZ domain stock per experiment
(concentration 0.5–2 mM).
3.3 Binding Assay
Parameters and Data
Collection
Below are the experimental parameters defined in the FluorEssence
V3.8 software. Anisotropy data is collected after each addition of
PDZ protein.
1. Experiment type: anisotropy.
2. Temperature: 25
C (or other desired temperature).
3. Number of data points is ~29 (see Note 11).
4. After adding the appropriate volume of PDZ domain, gently
mix the sample using a micropipette to avoid out-gassing followed by the measurement of fluorescence anisotropy (three
measurements are taken and averaged). A typical titration
experiment uses the following PDZ domain concentrations
and volumes per titration step.
5. Data point 1: the initial background measurement without any
PDZ domain. This serves as the baseline control.
6. Data point 2 and 3: add 3 μL of 100-fold diluted stock PDZ
domain (concentration 5–20 μM) at each step.
7. Data point 4 and 5: add 5 μL of 100-fold diluted stock PDZ
domain (concentration 5À20 μM) at each step.
8. Data point 6: add 10 μL of 100-fold diluted stock PDZ domain
(concentration 5–20 μM).
9. Data point 7 and 8: add 5 μL of tenfold diluted stock PDZ
domain (concentration 50–200 μM) at each step.
10. Data point 9 and 10: add 3 μL of PDZ domain stock (concentration 0.5–2 mM) at each step.
11. Data point 11 and 12: add 5 μL of PDZ domain stock (concentration 0.5–2 mM) at each step.
12. Data point 13 and 14: add 10 μL of PDZ domain stock
(concentration 0.5–2 mM) at each step.
PDZ Domain Binding Thermodynamics
141
introducing bubbles (see Note 9).
6. Cover the cuvette to prevent introducing dust particles and to
minimize the dansyl-fluorophore light exposure (see Note 10).
3.2.2 Preparation of PDZ
Domain Dilution Stock
Solutions
All the PDZ domain diluted solutions described below should be
prepared in advance and on ice.
1. Prepare 26 μL of a 100-fold diluted PDZ domain stock per
experiment (concentration 5–20 μM).
2. Prepare 10 μL of a tenfold diluted PDZ domain stock per
experiment (concentration 50–200 μM).
3. Prepare 656 μL PDZ domain stock per experiment
(concentration 0.5–2 mM).
3.3 Binding Assay
Parameters and Data
Collection
Below are the experimental parameters defined in the FluorEssence
V3.8 software. Anisotropy data is collected after each addition of
PDZ protein.
1. Experiment type: anisotropy.
2. Temperature: 25
C (or other desired temperature).
3. Number of data points is ~29 (see Note 11).
4. After adding the appropriate volume of PDZ domain, gently
mix the sample using a micropipette to avoid out-gassing followed by the measurement of fluorescence anisotropy (three
measurements are taken and averaged). A typical titration
experiment uses the following PDZ domain concentrations
and volumes per titration step.
5. Data point 1: the initial background measurement without any
PDZ domain. This serves as the baseline control.
6. Data point 2 and 3: add 3 μL of 100-fold diluted stock PDZ
domain (concentration 5–20 μM) at each step.
7. Data point 4 and 5: add 5 μL of 100-fold diluted stock PDZ
domain (concentration 5À20 μM) at each step.
8. Data point 6: add 10 μL of 100-fold diluted stock PDZ domain
(concentration 5–20 μM).
9. Data point 7 and 8: add 5 μL of tenfold diluted stock PDZ
domain (concentration 50–200 μM) at each step.
10. Data point 9 and 10: add 3 μL of PDZ domain stock (concentration 0.5–2 mM) at each step.
11. Data point 11 and 12: add 5 μL of PDZ domain stock (concentration 0.5–2 mM) at each step.
12. Data point 13 and 14: add 10 μL of PDZ domain stock
(concentration 0.5–2 mM) at each step.
PDZ Domain Binding Thermodynamics
141