1. Lyophilized peptides are solubilized at 10 mM according to the
manufacturer instructions and physicochemical properties of
the peptide sequences. Ideally, only water should be used, but
addition of 10 mM NaOH or HCl can help in solubilization of
peptides with acidic or basic properties, respectively. Use
DMSO in case of peptides with hydrophobic sequences. Solutions that contained primary amines are prohibited as primary
amines will interfere with cross-link reactions. Keep at 4
C
until use and then aliquot and store at - 80
C.
2. Using a 1ml tip cut at its extremity transfer the equivalent of
0.5ml of packed NHS activated Sepharose 4 Fast Flow beads in
15 mL-conical tubes containing 2–3 mL of cold isopropanol.
3. Centrifuge at 800 Â g for 5 min, 4
C, check the volume
corresponding to sedimented beads and adjust by adding
beads if needed. When 0.5 mL of packed beads is reached,
store the microfuge tube at 4
C until use.
4. Mix 100 μL of solubilized peptides with 900 μL MES buffer, in
a 1.5-mL microfuge tube and keep at 4
C.
5. Repeat this operation for all peptides that should be crosslinked.
6. Do an extra condition without peptide corresponding to control beads alone.
7. Keep microfuge tubes containing diluted peptides and control
at 4
C.
8. The next steps should be done extremely rapidly and at 4
C as
NHS beads will be active as soon as it will be in aqueous
solutions.
9. For all tubes, remove the isopropanol supernatant above the
beads.
10. Add 10 mL of cold 1 mM HCl.
11. Mix rapidly but gently without vortexing to resuspend the
beads.
12. Centrifuge at 800 Â g for 5 min, 4
C.
13. Remove the supernatant.
14. Transfer the peptide or control solutions made in steps 4–6 to
the beads prepared in step 2.
15. Mix rapidly.
16. Let on wheels overnight (see Note 16).
ä
Fig. 4 (continued) separated on 4–12% NuPAGE
®
acrylamide gel and silver stained. (b) Proteins associated to
TANC1 peptides and identified by MS. Volcano plots showing differential Log2 (peptide intensity) levels (x axis)
and –Log(p-value) (y axis) for TANC1 versus TANC1ΔPDZBM
Identification of Associated PDZ Proteins
27
manufacturer instructions and physicochemical properties of
the peptide sequences. Ideally, only water should be used, but
addition of 10 mM NaOH or HCl can help in solubilization of
peptides with acidic or basic properties, respectively. Use
DMSO in case of peptides with hydrophobic sequences. Solutions that contained primary amines are prohibited as primary
amines will interfere with cross-link reactions. Keep at 4
C
until use and then aliquot and store at - 80
C.
2. Using a 1ml tip cut at its extremity transfer the equivalent of
0.5ml of packed NHS activated Sepharose 4 Fast Flow beads in
15 mL-conical tubes containing 2–3 mL of cold isopropanol.
3. Centrifuge at 800 Â g for 5 min, 4
C, check the volume
corresponding to sedimented beads and adjust by adding
beads if needed. When 0.5 mL of packed beads is reached,
store the microfuge tube at 4
C until use.
4. Mix 100 μL of solubilized peptides with 900 μL MES buffer, in
a 1.5-mL microfuge tube and keep at 4
C.
5. Repeat this operation for all peptides that should be crosslinked.
6. Do an extra condition without peptide corresponding to control beads alone.
7. Keep microfuge tubes containing diluted peptides and control
at 4
C.
8. The next steps should be done extremely rapidly and at 4
C as
NHS beads will be active as soon as it will be in aqueous
solutions.
9. For all tubes, remove the isopropanol supernatant above the
beads.
10. Add 10 mL of cold 1 mM HCl.
11. Mix rapidly but gently without vortexing to resuspend the
beads.
12. Centrifuge at 800 Â g for 5 min, 4
C.
13. Remove the supernatant.
14. Transfer the peptide or control solutions made in steps 4–6 to
the beads prepared in step 2.
15. Mix rapidly.
16. Let on wheels overnight (see Note 16).
ä
Fig. 4 (continued) separated on 4–12% NuPAGE
®
acrylamide gel and silver stained. (b) Proteins associated to
TANC1 peptides and identified by MS. Volcano plots showing differential Log2 (peptide intensity) levels (x axis)
and –Log(p-value) (y axis) for TANC1 versus TANC1ΔPDZBM
Identification of Associated PDZ Proteins
27