7. Elute the peptide by a linear gradient of Eluent B (see Note 2).
8. Freeze the fractions at À80
C, then lyophilize them overnight.
9. Check the molecular mass of the relevant fraction by mass
spectrometry (important, see Note 3). Prepare the stock solution for FP as described above and store at À20
C.
3.2 Fluorescence
Polarization
(FP) to Study
the Effects
of Phosphorylation
Fluorescence polarization (FP) is a fast and high-throughput
method that is capable of measure steady-state dissociation constants of PPIs [20]. FP measurements can be performed in a direct
and competitive way (Fig. 2). In a direct assay, the formation of the
labeled (phospho/unphospho) peptide–PDZ domain complex is
monitored as a function of the PDZ domain concentration. In a
competitive assay, the dissociation of the labeled peptide–PDZ
domain complex is investigated as a function of the unlabeled
PDZ partner concentration [21]. The advantage of the former is
its simplicity; however, the fluorescent moiety can affect the affinity
of the peptide as the competitive assay benefits from the binding of
the competitors are not biased by the fluorescent moiety
[22]. Phosphorylation patterns can be studied by FP method in a
high-throughput way, quickly providing reliable steady-state dissociation constant for the PDZ–phospho/unphosphoPBM interaction by applying only small amounts of PDZ/PBM
compounds [14].
3.2.1 Direct Assay
1. Prepare 500 μl of tracer solution #1 (labeled phospho/unphospho RSK1-PBM 729–735 ) by adding 2.5 μl of labeled peptide
solution to 497.5 μl of buffer B to reach a 50 nM final
concentration.
2. Prepare 100 μl of tracer solution #2 (labeled phospho/unphospho RSK1-PBM 729–735 ) by adding 5 μl of labeled peptide
solution to 495 μl of buffer B to reach a 100 nM final
concentration.
3. Distribute 50 μl of tracer solution #2 into the first well and
50 μl of tracer solution #1 in the next seven vials in a 96-well
mixing plate (see Note 4).
4. Dilute the PDZ stock solutions in buffer B to 200 μM final
concentration by mixing 20 μl PDZ stock solution with 80 μl
buffer B (assuming 1 mM PDZ stock solutions).
5. Mix 50 μl of the PDZ domain solution with tracer solution #2
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.
184
Ma ´ rton A. Simon and La ´ szlo ´ Nyitray
8. Freeze the fractions at À80
C, then lyophilize them overnight.
9. Check the molecular mass of the relevant fraction by mass
spectrometry (important, see Note 3). Prepare the stock solution for FP as described above and store at À20
C.
3.2 Fluorescence
Polarization
(FP) to Study
the Effects
of Phosphorylation
Fluorescence polarization (FP) is a fast and high-throughput
method that is capable of measure steady-state dissociation constants of PPIs [20]. FP measurements can be performed in a direct
and competitive way (Fig. 2). In a direct assay, the formation of the
labeled (phospho/unphospho) peptide–PDZ domain complex is
monitored as a function of the PDZ domain concentration. In a
competitive assay, the dissociation of the labeled peptide–PDZ
domain complex is investigated as a function of the unlabeled
PDZ partner concentration [21]. The advantage of the former is
its simplicity; however, the fluorescent moiety can affect the affinity
of the peptide as the competitive assay benefits from the binding of
the competitors are not biased by the fluorescent moiety
[22]. Phosphorylation patterns can be studied by FP method in a
high-throughput way, quickly providing reliable steady-state dissociation constant for the PDZ–phospho/unphosphoPBM interaction by applying only small amounts of PDZ/PBM
compounds [14].
3.2.1 Direct Assay
1. Prepare 500 μl of tracer solution #1 (labeled phospho/unphospho RSK1-PBM 729–735 ) by adding 2.5 μl of labeled peptide
solution to 497.5 μl of buffer B to reach a 50 nM final
concentration.
2. Prepare 100 μl of tracer solution #2 (labeled phospho/unphospho RSK1-PBM 729–735 ) by adding 5 μl of labeled peptide
solution to 495 μl of buffer B to reach a 100 nM final
concentration.
3. Distribute 50 μl of tracer solution #2 into the first well and
50 μl of tracer solution #1 in the next seven vials in a 96-well
mixing plate (see Note 4).
4. Dilute the PDZ stock solutions in buffer B to 200 μM final
concentration by mixing 20 μl PDZ stock solution with 80 μl
buffer B (assuming 1 mM PDZ stock solutions).
5. Mix 50 μl of the PDZ domain solution with tracer solution #2
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.
184
Ma ´ rton A. Simon and La ´ szlo ´ Nyitray