labeled peptide to 50 nM, the amount of PDZ domain can be
calculated based on the direct FP measurement.
1. Prepare 500 μl of tracer solution 3# (labeled phospho/unphospho RSK1-PBM 729–735 and PDZ domain) by adding 2.5 μl of
labeled peptide solution and V PDZ μl to 497.5 - V PDZ μl of
buffer B to reach a 50 nM final concentration for the labeled
peptide which saturates the PDZ domain in 60–80%.
2. Prepare 100 μl of tracer solution 4# (labeled phospho/unphospho RSK1-PBM 729–735 and PDZ domain) by adding 5 μl of
labeled peptide solution and 2V PDZ μl to 495 - 2V PDZ μl of
buffer B to reach a 100 nM final concentration for the labeled
peptide which saturates the PDZ domain in 60–80%.
3. Distribute 50 μl of tracer solution #4 into the first well and
50 μl of tracer solution #3 in the next seven vials in a 98-well
mixing plate (see Note 4).
4. Dilute the unlabeled RSK fragment stock solutions in buffer B
to 200 μM final concentration by mixing 20 μl RSK stock
solution/RSK recombinant fragment stock solution with
80 μl buffer B (assuming 1 mM unlabeled recombinant fragment stock solutions).
5. Mix 50 μl of the unlabeled recombinant fragment solution with
tracer solution #4 in the first well to get 100 μM final concentration (see Note 4).
6. Perform a twofold serial dilution in the following way: pipet
50 μl solution from the very first well to the next well, mix it
carefully, then pipet 50 μl from this one to the next well. Repeat
this, until you reach the one, prior to the last well. Do not
dilute the last well.
7. Pipet 15–15 μl from the last well (which is not diluted) of the
serial dilution to three horizontal wells of the black 384-well
plate, then repeat this procedure with all wells of the serial
dilution. This way, 3 Â 8 (24) wells’ fluorescence is measured
by the plate reader.
8. Analyze the data using Origin software (or other data analyzer
software package) applying competitive binding models [23]
(see Note 5).
3.3 Isothermal
Titration Calorimetry
(ITC) to Study
the Effects
of Phosphorylation
Besides validating steady-state K d values, ITC measurements provide additional thermodynamic parameters of the reaction (such as
the change in enthalpy or entropy), moreover it is a label-free
method to detect interactions [24]. As drawbacks, ITC measurements are low-throughput and require large amount of material
(compared to FP assays). Moreover very weak interactions cannot
be characterized reliably. Though PDZ domain–RSK1 interactions
fall into the higher micromolar range, the phosphorylation induced
186
Ma ´ rton A. Simon and La ´ szlo ´ Nyitray
calculated based on the direct FP measurement.
1. Prepare 500 μl of tracer solution 3# (labeled phospho/unphospho RSK1-PBM 729–735 and PDZ domain) by adding 2.5 μl of
labeled peptide solution and V PDZ μl to 497.5 - V PDZ μl of
buffer B to reach a 50 nM final concentration for the labeled
peptide which saturates the PDZ domain in 60–80%.
2. Prepare 100 μl of tracer solution 4# (labeled phospho/unphospho RSK1-PBM 729–735 and PDZ domain) by adding 5 μl of
labeled peptide solution and 2V PDZ μl to 495 - 2V PDZ μl of
buffer B to reach a 100 nM final concentration for the labeled
peptide which saturates the PDZ domain in 60–80%.
3. Distribute 50 μl of tracer solution #4 into the first well and
50 μl of tracer solution #3 in the next seven vials in a 98-well
mixing plate (see Note 4).
4. Dilute the unlabeled RSK fragment stock solutions in buffer B
to 200 μM final concentration by mixing 20 μl RSK stock
solution/RSK recombinant fragment stock solution with
80 μl buffer B (assuming 1 mM unlabeled recombinant fragment stock solutions).
5. Mix 50 μl of the unlabeled recombinant fragment solution with
tracer solution #4 in the first well to get 100 μM final concentration (see Note 4).
6. Perform a twofold serial dilution in the following way: pipet
50 μl solution from the very first well to the next well, mix it
carefully, then pipet 50 μl from this one to the next well. Repeat
this, until you reach the one, prior to the last well. Do not
dilute the last well.
7. Pipet 15–15 μl from the last well (which is not diluted) of the
serial dilution to three horizontal wells of the black 384-well
plate, then repeat this procedure with all wells of the serial
dilution. This way, 3 Â 8 (24) wells’ fluorescence is measured
by the plate reader.
8. Analyze the data using Origin software (or other data analyzer
software package) applying competitive binding models [23]
(see Note 5).
3.3 Isothermal
Titration Calorimetry
(ITC) to Study
the Effects
of Phosphorylation
Besides validating steady-state K d values, ITC measurements provide additional thermodynamic parameters of the reaction (such as
the change in enthalpy or entropy), moreover it is a label-free
method to detect interactions [24]. As drawbacks, ITC measurements are low-throughput and require large amount of material
(compared to FP assays). Moreover very weak interactions cannot
be characterized reliably. Though PDZ domain–RSK1 interactions
fall into the higher micromolar range, the phosphorylation induced
186
Ma ´ rton A. Simon and La ´ szlo ´ Nyitray