2.2 Fluorescence
Polarization
1. Fluorescence polarization (FP) buffer: 50 mM sodium phosphate (NaPi) pH 7.5. Dissolve 6.4 g of Na 2 HPO 4 and 1.58 g
of NaH 2 PO 4 in 800 mL distilled water. Adjust the pH to 7.5
by adding either 0.1 M NaOH or 0.1 M HCl. Fill up to a total
volume of 1 L with distilled water.
2. Cy5 conjugated peptide probe: prepared as 5 μM stock in FP
buffer
3. 384-well plate, black flat-bottom
4. Analytical pipette channels, with low protein binding pipette
tips
5. 10Â bovine serum albumin (BSA) stock: Dissolve 1 g BSA in
10 mL FP buffer
2.3 Isothermal
Calorimetry
1. PBS buffer (see Subheading 2.1). Buffer is degassed and
filtered.
2. Protein and peptide samples dissolved in the same exact PBS
buffer.
2.4 Pull-Down
of Nonischemic Brain
Lysates
1. Biotin-labeled compound (see Subheading 3.1 and Note 1).
2. 10–12-week old C57BL/6J
Babr mice (Charles River Laboratories, Margate, UK).
3. Streptavidin-conjugated resin (Thermo Scientific, Loughborough, UK).
4. Immobilization/Homogenization buffer: 1 M Tris–HCl
pH 7.0 containing 0.05% sodium deoxycholate. Dissolve
121.14 g of Tris-base in 800 mL distilled water. Adjust the
pH to 7.0 by adding 0.1 M HCl. Fill up to a total volume of 1 L
with distilled water and add 0.5 g sodium deoxycholate.
5. Sample Buffer: 4% sodium dodecyl sulfate (SDS), 20% glycerol,
10% 2-mercaptoethanol, 0.004% bromophenol blue, and
0.125 M Tris–HCl pH 6.8. Dissolve 0.4 g SDS, 2 mL glycerol,
1.25 mL of 1 M Tris–HCl (pH 6.8), and 20 mg bromophenol
blue in 10.7 mL distilled water, or purchase premixed sample
buffer (Sigma-Aldrich Corp., St.Louis, USA).
6. Pierce™ bicinchoninic acid assay (BCA) protein quantitation
kit (Thermo Scientific, Loughborough, UK).
7. Pierce™ ECL Western Blotting Substrate (Thermo Scientific,
Loughborough, UK).
8. β-mercaptoethanol.
9. PSD-95 monoclonal antibody (cat. No. MA1-045, Thermo
Fisher, Loughborough, UK).
10. PSD-93 monoclonal antibody (cat. No. 75-057; NeuroMab,
Davis, USA).
PDZ Domain Peptide Inhibitors
161
Polarization
1. Fluorescence polarization (FP) buffer: 50 mM sodium phosphate (NaPi) pH 7.5. Dissolve 6.4 g of Na 2 HPO 4 and 1.58 g
of NaH 2 PO 4 in 800 mL distilled water. Adjust the pH to 7.5
by adding either 0.1 M NaOH or 0.1 M HCl. Fill up to a total
volume of 1 L with distilled water.
2. Cy5 conjugated peptide probe: prepared as 5 μM stock in FP
buffer
3. 384-well plate, black flat-bottom
4. Analytical pipette channels, with low protein binding pipette
tips
5. 10Â bovine serum albumin (BSA) stock: Dissolve 1 g BSA in
10 mL FP buffer
2.3 Isothermal
Calorimetry
1. PBS buffer (see Subheading 2.1). Buffer is degassed and
filtered.
2. Protein and peptide samples dissolved in the same exact PBS
buffer.
2.4 Pull-Down
of Nonischemic Brain
Lysates
1. Biotin-labeled compound (see Subheading 3.1 and Note 1).
2. 10–12-week old C57BL/6J
Babr mice (Charles River Laboratories, Margate, UK).
3. Streptavidin-conjugated resin (Thermo Scientific, Loughborough, UK).
4. Immobilization/Homogenization buffer: 1 M Tris–HCl
pH 7.0 containing 0.05% sodium deoxycholate. Dissolve
121.14 g of Tris-base in 800 mL distilled water. Adjust the
pH to 7.0 by adding 0.1 M HCl. Fill up to a total volume of 1 L
with distilled water and add 0.5 g sodium deoxycholate.
5. Sample Buffer: 4% sodium dodecyl sulfate (SDS), 20% glycerol,
10% 2-mercaptoethanol, 0.004% bromophenol blue, and
0.125 M Tris–HCl pH 6.8. Dissolve 0.4 g SDS, 2 mL glycerol,
1.25 mL of 1 M Tris–HCl (pH 6.8), and 20 mg bromophenol
blue in 10.7 mL distilled water, or purchase premixed sample
buffer (Sigma-Aldrich Corp., St.Louis, USA).
6. Pierce™ bicinchoninic acid assay (BCA) protein quantitation
kit (Thermo Scientific, Loughborough, UK).
7. Pierce™ ECL Western Blotting Substrate (Thermo Scientific,
Loughborough, UK).
8. β-mercaptoethanol.
9. PSD-95 monoclonal antibody (cat. No. MA1-045, Thermo
Fisher, Loughborough, UK).
10. PSD-93 monoclonal antibody (cat. No. 75-057; NeuroMab,
Davis, USA).
PDZ Domain Peptide Inhibitors
161