6. Eluent B: 0.09% (v/v) TFA in acetonitrile. Add 500 ml HPLC
grade acetonitrile into a graduated cylinder then supplement it
with 450 μl TFA.
7. Jupiter 300 C5 RP-HPLC column (Phenomenex).
8. 0.45 μm pore size syringe filters.
2.2 Fluorescence
Polarization (FP)
1. FP buffer (buffer B): 150 mM NaCl, 100 μM TCEP, 0.01%
Tween 20, 40 mM HEPES–NaOH pH 7.5. Dissolve 0.88 g
NaCl and 0.95 g HEPES in 95 ml Milli-Q water. Add 20 μl
0.5 M TCEP solution and 100 μl 10% Tween 20. Adjust the
pH to 7.5 with sodium hydroxide solution, then fill it to
100 ml with Milli-Q water. Store at À20
C in 10 ml aliquots.
2. 10 μM labeled peptides: Prepare 1 mM stock solutions of the
chemically synthesized and carboxyfluorescein-labeled phospho/unphospho RSK1 729–735 peptide by dissolving approximately 0.7 mg phospho/unphospho peptide in 0.5 ml DMSO.
Determine the concentration of the stock solutions by measuring the absorbance of the 5(6)-carboxyfluorescein at 495 nm
(ε coeff ¼ 82,000 M
À1 cm
À1
). Dilute the stock solutions in
buffer B to 10 μM. Store at À20
C in 100 μl aliquots.
3. 1 mM unlabeled peptide stock solution: To prepare 1 mM
stock
solutions
of
unlabeled
phospho/unphospho
RSK1 683–735 , dissolve approximately 7.2 mg of lyophilized
peptides in 1 ml Milli-Q water. Determine the concentration
by measuring the absorbance of the Tyr residue at 280 nm
(ε coeff ¼ 1490 M
À1 cm
À1 ). Store at À20
C in 100 μl aliquots.
4. PDZ domains are kept as stock solutions of at least 1 mM. Pure
PDZ domains can be obtained through a tandem purification
approach using Ni
2+ and MBP affinity chromatography after
bacterial expression described elsewhere [9]. Upon mixing
with 20% glycerol, 100 μM TCEP (reducing agent) and adjusting its final concentration to 1 mM, the recombinant PDZ
domains are distributed in 100 μl aliquots that are subsequently
frozen in liquid nitrogen and stored at À80
C.
5. Black 384-well nonbinding microplates.
6. Plate reader.
2.3 Isothermal
Titration
Calorimetry (ITC)
1. ITC buffer (Buffer C): 150 mM NaCl, 50 nM TCEP, 20 mM
HEPES–NaOH pH 7.5. Dissolve 4.76 g HEPES and 8.77 g
NaCl in 950 ml deionized water. Add 100 μl 0.5 M TCEP and
adjust the pH to 7.5 with sodium hydroxide solution, then
complete the volume to 1 l with Milli-Q water.
2. 1 mM PDZ domain stock solution. Described above previously
in item 4 Subheading 2.2.
182
Ma ´ rton A. Simon and La ´ szlo ´ Nyitray
grade acetonitrile into a graduated cylinder then supplement it
with 450 μl TFA.
7. Jupiter 300 C5 RP-HPLC column (Phenomenex).
8. 0.45 μm pore size syringe filters.
2.2 Fluorescence
Polarization (FP)
1. FP buffer (buffer B): 150 mM NaCl, 100 μM TCEP, 0.01%
Tween 20, 40 mM HEPES–NaOH pH 7.5. Dissolve 0.88 g
NaCl and 0.95 g HEPES in 95 ml Milli-Q water. Add 20 μl
0.5 M TCEP solution and 100 μl 10% Tween 20. Adjust the
pH to 7.5 with sodium hydroxide solution, then fill it to
100 ml with Milli-Q water. Store at À20
C in 10 ml aliquots.
2. 10 μM labeled peptides: Prepare 1 mM stock solutions of the
chemically synthesized and carboxyfluorescein-labeled phospho/unphospho RSK1 729–735 peptide by dissolving approximately 0.7 mg phospho/unphospho peptide in 0.5 ml DMSO.
Determine the concentration of the stock solutions by measuring the absorbance of the 5(6)-carboxyfluorescein at 495 nm
(ε coeff ¼ 82,000 M
À1 cm
À1
). Dilute the stock solutions in
buffer B to 10 μM. Store at À20
C in 100 μl aliquots.
3. 1 mM unlabeled peptide stock solution: To prepare 1 mM
stock
solutions
of
unlabeled
phospho/unphospho
RSK1 683–735 , dissolve approximately 7.2 mg of lyophilized
peptides in 1 ml Milli-Q water. Determine the concentration
by measuring the absorbance of the Tyr residue at 280 nm
(ε coeff ¼ 1490 M
À1 cm
À1 ). Store at À20
C in 100 μl aliquots.
4. PDZ domains are kept as stock solutions of at least 1 mM. Pure
PDZ domains can be obtained through a tandem purification
approach using Ni
2+ and MBP affinity chromatography after
bacterial expression described elsewhere [9]. Upon mixing
with 20% glycerol, 100 μM TCEP (reducing agent) and adjusting its final concentration to 1 mM, the recombinant PDZ
domains are distributed in 100 μl aliquots that are subsequently
frozen in liquid nitrogen and stored at À80
C.
5. Black 384-well nonbinding microplates.
6. Plate reader.
2.3 Isothermal
Titration
Calorimetry (ITC)
1. ITC buffer (Buffer C): 150 mM NaCl, 50 nM TCEP, 20 mM
HEPES–NaOH pH 7.5. Dissolve 4.76 g HEPES and 8.77 g
NaCl in 950 ml deionized water. Add 100 μl 0.5 M TCEP and
adjust the pH to 7.5 with sodium hydroxide solution, then
complete the volume to 1 l with Milli-Q water.
2. 1 mM PDZ domain stock solution. Described above previously
in item 4 Subheading 2.2.
182
Ma ´ rton A. Simon and La ´ szlo ´ Nyitray