2. Biotin-labeled PSD-95 peptide inhibitors were bound to
streptavidin-conjugated resin by incubating 2.5 mM of each
peptide with 120 μL of resin in immobilization buffer (see Note
17).
3. Resin and peptide solution were incubated for 3 h at room
temperature before the peptide supernatant was removed and
the resin was washed with immobilization buffer and stored at
4
C.
4. The pull-down is performed using extracts from forebrains of
10–12-week-old C57BL/6J
Babr mice (Charles River Laboratories, Margate, UK).
5. Homogenization of the tissue in homogenization buffer by a
tissue grinder (Kimble Chase, Vineland, USA) (see Note 18).
6. Incubate the lysate for 1 h on ice.
7. Spin down at 20,000 rpm for 20 min with an ultracentrifuge at
4
C and collect the supernatant.
8. Measure the protein concentration of the lysate using Pierce™
bicinchoninic acid assay (BCA) protein quantitation kit and
dilute the samples to 2 μg μL
À1
9. Peptide-coupled resin was aliquoted into 5 Â 20 μL samples
before adding protein lysate. The lysate was added at different
protein-to-resin ratios: 1:10, 1:50; 1:100, 1:500; 1:1000 and
rotated at 4
C overnight.
10. Spin down the samples and collect the pellet from each condition, and discard the remaining supernatant.
11. Wash the resin thoroughly with homogenization buffer.
12. Elute the proteins by heating to 70
C for 30 min in sample
buffer.
13. Samples were run on SDS-PAGE gels and blots were probed
for MAGUK proteins using primary antibodies against
PSD-95, PSD-93, SAP-97, and SAP-102 (Fig. 5).
14. Incubate the SDS-PAGE with the primary antibody overnight
and subsequently apply horseradish peroxidase (HRP) secondary antibodies for 1 h at room temperature.
15. Visualize the blots with Pierce™ ECL Western Blotting Substrate on a GE Healthcare Life Science ImageQuant 4000
imager.
16. For negative control see Note 19.
3.4.2 Blood Plasma
stability Assay
An important measurement in the early-stage drug development
process is the stability of the compound in plasma. This assay is
especially important for peptide-based drugs as peptides and proteins are degraded by proteases.
PDZ Domain Peptide Inhibitors
171
streptavidin-conjugated resin by incubating 2.5 mM of each
peptide with 120 μL of resin in immobilization buffer (see Note
17).
3. Resin and peptide solution were incubated for 3 h at room
temperature before the peptide supernatant was removed and
the resin was washed with immobilization buffer and stored at
4
C.
4. The pull-down is performed using extracts from forebrains of
10–12-week-old C57BL/6J
Babr mice (Charles River Laboratories, Margate, UK).
5. Homogenization of the tissue in homogenization buffer by a
tissue grinder (Kimble Chase, Vineland, USA) (see Note 18).
6. Incubate the lysate for 1 h on ice.
7. Spin down at 20,000 rpm for 20 min with an ultracentrifuge at
4
C and collect the supernatant.
8. Measure the protein concentration of the lysate using Pierce™
bicinchoninic acid assay (BCA) protein quantitation kit and
dilute the samples to 2 μg μL
À1
9. Peptide-coupled resin was aliquoted into 5 Â 20 μL samples
before adding protein lysate. The lysate was added at different
protein-to-resin ratios: 1:10, 1:50; 1:100, 1:500; 1:1000 and
rotated at 4
C overnight.
10. Spin down the samples and collect the pellet from each condition, and discard the remaining supernatant.
11. Wash the resin thoroughly with homogenization buffer.
12. Elute the proteins by heating to 70
C for 30 min in sample
buffer.
13. Samples were run on SDS-PAGE gels and blots were probed
for MAGUK proteins using primary antibodies against
PSD-95, PSD-93, SAP-97, and SAP-102 (Fig. 5).
14. Incubate the SDS-PAGE with the primary antibody overnight
and subsequently apply horseradish peroxidase (HRP) secondary antibodies for 1 h at room temperature.
15. Visualize the blots with Pierce™ ECL Western Blotting Substrate on a GE Healthcare Life Science ImageQuant 4000
imager.
16. For negative control see Note 19.
3.4.2 Blood Plasma
stability Assay
An important measurement in the early-stage drug development
process is the stability of the compound in plasma. This assay is
especially important for peptide-based drugs as peptides and proteins are degraded by proteases.
PDZ Domain Peptide Inhibitors
171