13. Make a 100-fold dilution of the EGF stock in Milli-Q water to
obtain a 1.35 μg/ml EGF solution.
14. Stimulate the cells of 3 wells with 10 μl of 1.35 μg/ml EGF
solution (100 ng/ml final concentration of EGF) while treating the cells of 3 control wells with Milli-Q water (see Note
17).
15. Start the measurement for 1 h recording the luminescence
signal for each well as many times as possible.
16. Evaluate the data by normalizing the signal. For a steady state
measurement, values are normalized to the luminescence of the
wild-type protein. For a dynamic measurement, the values are
normalized to the average signal decay of the luminescence of
unstimulated wells.
4 Notes
1. The kinase of interest is susceptible to precipitation as the pH
has impact on the stability of the kinase, therefore do not forget
to adjust the pH of the reaction mixture to 7.5 before adding
the kinase itself.
2. Peptides typically elute between 30% and 45% Eluent B.
3. Note that the phosphorylation reaction sometimes does not go
very well due to the characteristics of the applied kinase. Do not
forget to check your freshly prepared fraction by mass
spectrometry!
4. As a result, the concentration of the labeled peptide (and the
PDZ domain in competitive measurements) will be equal in all
wells. If well-concentrated stocks solution of PDZ domain and
unlabeled recombinant fragment are available (e.g., 5 mM),
then One can add the PDZ to tracer solution #1 (e.g., 5 μl
5 mM PDZ + 95 μl tracer solution #1 followed by the twofold
dilution step) in direct titration and the unlabeled RSK fragment to tracer solution #3 (e.g., 5 μl 5 mM unlabeled
RSK + 95 μl tracer solution #3 followed by the twofold dilution
step) in competitive titration.
5. An automated, unbiased fitting program called ProFit is available, which can be found on GitHub (https://github.com/
GoglG/ProFit) and is described in reference [26].
6. For the competition assay, one can apply less saturated complexes in cases of weaker interactions if the difference between
the base polarization and the polarization at the given saturation is large enough (>50 mP).
7. For a competitive assay, a direct FP measurement is also
required to be analyzed. Furthermore, to avoid experimental
Regulation of RSK1-PDZ Domain Interactions
189
obtain a 1.35 μg/ml EGF solution.
14. Stimulate the cells of 3 wells with 10 μl of 1.35 μg/ml EGF
solution (100 ng/ml final concentration of EGF) while treating the cells of 3 control wells with Milli-Q water (see Note
17).
15. Start the measurement for 1 h recording the luminescence
signal for each well as many times as possible.
16. Evaluate the data by normalizing the signal. For a steady state
measurement, values are normalized to the luminescence of the
wild-type protein. For a dynamic measurement, the values are
normalized to the average signal decay of the luminescence of
unstimulated wells.
4 Notes
1. The kinase of interest is susceptible to precipitation as the pH
has impact on the stability of the kinase, therefore do not forget
to adjust the pH of the reaction mixture to 7.5 before adding
the kinase itself.
2. Peptides typically elute between 30% and 45% Eluent B.
3. Note that the phosphorylation reaction sometimes does not go
very well due to the characteristics of the applied kinase. Do not
forget to check your freshly prepared fraction by mass
spectrometry!
4. As a result, the concentration of the labeled peptide (and the
PDZ domain in competitive measurements) will be equal in all
wells. If well-concentrated stocks solution of PDZ domain and
unlabeled recombinant fragment are available (e.g., 5 mM),
then One can add the PDZ to tracer solution #1 (e.g., 5 μl
5 mM PDZ + 95 μl tracer solution #1 followed by the twofold
dilution step) in direct titration and the unlabeled RSK fragment to tracer solution #3 (e.g., 5 μl 5 mM unlabeled
RSK + 95 μl tracer solution #3 followed by the twofold dilution
step) in competitive titration.
5. An automated, unbiased fitting program called ProFit is available, which can be found on GitHub (https://github.com/
GoglG/ProFit) and is described in reference [26].
6. For the competition assay, one can apply less saturated complexes in cases of weaker interactions if the difference between
the base polarization and the polarization at the given saturation is large enough (>50 mP).
7. For a competitive assay, a direct FP measurement is also
required to be analyzed. Furthermore, to avoid experimental
Regulation of RSK1-PDZ Domain Interactions
189