17. Add 3 mL of ethanolamine to quench the reaction and keep on
wheels for 3 h.
18. Centrifuge at 800 Â g for 5 min, remove the supernatant, add
10 mL of Tris, and mix gently.
19. Centrifuge at 800 Â g, for 5 min, remove the supernatant, add
10 mL of Acetate, mix gently.
20. Repeat the last two steps twice.
21. Centrifuge at 800 Â g for 5 min, remove the supernatant, add
10 mL of PBS, and mix gently.
22. Repeat the last operation twice.
23. Centrifuge, remove the supernatant, and add to 0.5 mL dried
beads 4.5 mL of preservative solution for short-term storage
(see Note 17). Beads are ready to be used for peptide pulldown.
24. Just before pulldown, add 200 μL of the bead suspension,
corresponding to 20 μL of dried beads, to a clean microfuge
tube and centrifuge gently.
25. Equilibrate the beads once with 1 mL of lysis buffer used to
treat the cells or tissues.
26. Remove supernatants until 3–5 mm above the sedimented
beads to avoid drying.
3.2.3 Coupling
Biotinylated Peptides
with Streptavidin Beads
1. Streptavidin beads are always washed the day before the experiment with PBS to remove ethanol from the commercial stock.
2. Twenty microliters of dried beads will be used for each experimental point. Determine the volume you will need and add a
calculated extra volume to be sure to keep enough beads after
extensive washes.
3. To a 15-mL-conical tube, add 3–5 mL PBS.
4. Transfer the volume of streptavidin beads needed to the experiments to the 15 mL-conical tube and centrifuge at 800 Â g for
5 min at 4
C.
5. Discard the supernatant, check the bead volume, and adjust if
needed.
6. After the last centrifugation, add adequate volume of PBS to
obtain a 10% beads suspension (v/v).
7. Prepare adequate number of microfuge tubes (one per condition plus one extra microfuge tube corresponding to the control without peptide).
8. Add 500 μL PBS in a 1.5 mL microfuge tube and add 200 μL
of 10% beads suspension (20 μL packed beads) (v/v), centrifuge at 800 Â g for 5 min, and confirm the volume of packed
beads. Beads are now ready to use.
28
Avais M. Daulat et al.
wheels for 3 h.
18. Centrifuge at 800 Â g for 5 min, remove the supernatant, add
10 mL of Tris, and mix gently.
19. Centrifuge at 800 Â g, for 5 min, remove the supernatant, add
10 mL of Acetate, mix gently.
20. Repeat the last two steps twice.
21. Centrifuge at 800 Â g for 5 min, remove the supernatant, add
10 mL of PBS, and mix gently.
22. Repeat the last operation twice.
23. Centrifuge, remove the supernatant, and add to 0.5 mL dried
beads 4.5 mL of preservative solution for short-term storage
(see Note 17). Beads are ready to be used for peptide pulldown.
24. Just before pulldown, add 200 μL of the bead suspension,
corresponding to 20 μL of dried beads, to a clean microfuge
tube and centrifuge gently.
25. Equilibrate the beads once with 1 mL of lysis buffer used to
treat the cells or tissues.
26. Remove supernatants until 3–5 mm above the sedimented
beads to avoid drying.
3.2.3 Coupling
Biotinylated Peptides
with Streptavidin Beads
1. Streptavidin beads are always washed the day before the experiment with PBS to remove ethanol from the commercial stock.
2. Twenty microliters of dried beads will be used for each experimental point. Determine the volume you will need and add a
calculated extra volume to be sure to keep enough beads after
extensive washes.
3. To a 15-mL-conical tube, add 3–5 mL PBS.
4. Transfer the volume of streptavidin beads needed to the experiments to the 15 mL-conical tube and centrifuge at 800 Â g for
5 min at 4
C.
5. Discard the supernatant, check the bead volume, and adjust if
needed.
6. After the last centrifugation, add adequate volume of PBS to
obtain a 10% beads suspension (v/v).
7. Prepare adequate number of microfuge tubes (one per condition plus one extra microfuge tube corresponding to the control without peptide).
8. Add 500 μL PBS in a 1.5 mL microfuge tube and add 200 μL
of 10% beads suspension (20 μL packed beads) (v/v), centrifuge at 800 Â g for 5 min, and confirm the volume of packed
beads. Beads are now ready to use.
28
Avais M. Daulat et al.