9. Add for each condition 10 μL of 10 mM biotinylated peptides
or 10 μL of 10 mM biotin (control without peptide).
10. Incubate for 1 h on rotating wheels at 4
C.
11. Wash the beads twice with 1 mL PBS to remove unbound
peptides.
12. Discard the supernatant. Leave 3–5 mm of PBS above the
packed beads to avoid beads drying. Beads are now ready for
the peptide pulldown.
13. Add 20% ethanol (v/v). Peptide-coupled beads prepared with
this protocol can be stored for at least 1 year, meaning that a
large stock (1–2 mL Sepharose beads) can be prepared for
further use. In contrast, the half-life of streptavidin beads is
shorter. We thus recommend preparing extemporaneously
fresh streptavidin beads coupled with peptides.
3.2.4 Peptide Pulldown
1. Preparation of the lysates (see Notes 2 and 18).
2. Lysates should be used at 1–5 mg/mL of soluble proteins (see
Note 19).
3. Lysates should be precleared with control beads such as NHS
beads saturated with ethanolamine (as described in Subheading
3.2.2, step 6) or streptavidin beads (see Note 20), a ratio of
200 μL packed beads for 10 mL lysate is appropriate but can be
adjusted depending on the protein concentration of the lysates.
Preclearing is performed at 4
C on wheels for 45 min.
4. Centrifuge the lysates at 3000 Â g for 5 min to sediment the
beads.
5. Transfer gently the supernatants without any beads in another
tube compatible with a 16,000 Â g centrifugation.
6. Centrifuge the lysates at 16,000 Â g for 1 min to be sure to
eliminate extra beads or protein aggregates (see Note 21).
7. Transfer the supernatants to a new tube: the lysates are ready.
8. Transfer 1 mL of the lysates (1–5 mg/mL) in 1.5-mL-microfuge tubes containing beads bearing peptides or control beads.
9. Incubate for 2 h on wheels, at 4
C.
10. Wash the beads 3 to 5 times with 1 mL lysis buffer.
11. Add 20 μL 2Â LDS NuPAGE buffer and heat at 70
C for
10 min.
12. Centrifuge at 2000 Â g for 5 min.
13. Migrate 10% (2 or 3 μL) of the bead eluate on a NuPAGE
®
precast gel (4–12%) and perform a silver staining for quality
control of the experiment (Fig. 4a).
14. Store the remaining 90% at À20
C for the MS analysis.
Identification of Associated PDZ Proteins
29
or 10 μL of 10 mM biotin (control without peptide).
10. Incubate for 1 h on rotating wheels at 4
C.
11. Wash the beads twice with 1 mL PBS to remove unbound
peptides.
12. Discard the supernatant. Leave 3–5 mm of PBS above the
packed beads to avoid beads drying. Beads are now ready for
the peptide pulldown.
13. Add 20% ethanol (v/v). Peptide-coupled beads prepared with
this protocol can be stored for at least 1 year, meaning that a
large stock (1–2 mL Sepharose beads) can be prepared for
further use. In contrast, the half-life of streptavidin beads is
shorter. We thus recommend preparing extemporaneously
fresh streptavidin beads coupled with peptides.
3.2.4 Peptide Pulldown
1. Preparation of the lysates (see Notes 2 and 18).
2. Lysates should be used at 1–5 mg/mL of soluble proteins (see
Note 19).
3. Lysates should be precleared with control beads such as NHS
beads saturated with ethanolamine (as described in Subheading
3.2.2, step 6) or streptavidin beads (see Note 20), a ratio of
200 μL packed beads for 10 mL lysate is appropriate but can be
adjusted depending on the protein concentration of the lysates.
Preclearing is performed at 4
C on wheels for 45 min.
4. Centrifuge the lysates at 3000 Â g for 5 min to sediment the
beads.
5. Transfer gently the supernatants without any beads in another
tube compatible with a 16,000 Â g centrifugation.
6. Centrifuge the lysates at 16,000 Â g for 1 min to be sure to
eliminate extra beads or protein aggregates (see Note 21).
7. Transfer the supernatants to a new tube: the lysates are ready.
8. Transfer 1 mL of the lysates (1–5 mg/mL) in 1.5-mL-microfuge tubes containing beads bearing peptides or control beads.
9. Incubate for 2 h on wheels, at 4
C.
10. Wash the beads 3 to 5 times with 1 mL lysis buffer.
11. Add 20 μL 2Â LDS NuPAGE buffer and heat at 70
C for
10 min.
12. Centrifuge at 2000 Â g for 5 min.
13. Migrate 10% (2 or 3 μL) of the bead eluate on a NuPAGE
®
precast gel (4–12%) and perform a silver staining for quality
control of the experiment (Fig. 4a).
14. Store the remaining 90% at À20
C for the MS analysis.
Identification of Associated PDZ Proteins
29