2 Materials
Prepare all solutions using ultrapure water (prepared by purifying
deionized water, to attain a sensitivity of 18 MΩ-cm at 25
C) and
analytical grade reagents (standard organic solvents are not listed in
the Material section). Prepare and store all reagents at room temperature (unless indicated otherwise).
2.1 Peptide
Synthesis
1. Silicycle MiniBlock® 10 mL polypropylene disposable reaction
tubes (SiliCycle Inc., Que ´bec, Canada) or equivalent.
2. Wang resins preloaded with the C-terminal amino acid or
2-chlorotrityl chloride resin.
3. 9-Fluorenyl-methyloxycarbonyl (Fmoc)-protected amino acids
with standard side-chain protection groups.
4. 0.5 M 2-(1-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) or 0.5 M 2-(1H-7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU) in dimethylformamide (DMF). Dissolve
1.9 g HBTU or 1.9 g HATU in 10 mL DMF and store
solutions in light-protected bottles.
5. 20% (v/v) piperidine in DMF.
6. Kaiser test kit (Sigma-Aldrich Corp, St. Louis, USA).
7. Capping solution: 10% (v/v) acetic anhydride, 10% (v/v) N,Ndiisopropylethylamine (DIPEA), 80% (v/v) DMF. Mix 1 mL of
acetic anhydride, 1 mL of DIPEA in 8 mL of DMF. Prepare
solution fresh before use.
8. Cleavage cocktail: 80% (v/v) trifluoroacetic acid (TFA), 10%
(v/v) water, 10% (v/v) Triisopropylsilane (TIPS).
9. Ice-cold diethyl ether: store at À20
C until usage.
10. Cy5-Maleimide (GE Healthcare, Chicago, USA).
11. Polyethylene glycol (PEG)-diacid (Iris Biotech GmbH, Marktredwitz, Germany).
12. Biotin (lyophilized powder).
13. PBS buffer: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 ,
2 mM KH 2 PO 4 , pH 7.4. Dissolve 8 g NaCl, 200 mg KCl,
1.44 g Na 2 HPO 4 , and 240 mg KH 2 PO 4 in 800 mL distilled
water. Adjust the pH to 7.4 by adding either 0.1 M NaOH or
0.1 M HCl. Fill up to a total volume of 1 L with distilled water.
14. Triphenylphosphine (PPh 3 ).
15. Diisopropyl azodicarboxylate (DIAD).
16. Diazabicyclo[5.4.0]undec-7-ene (DBU).
17. 2,4,6-trimethylpyridine (Collidine).
160
Dominik J. Essig et al.
Prepare all solutions using ultrapure water (prepared by purifying
deionized water, to attain a sensitivity of 18 MΩ-cm at 25
C) and
analytical grade reagents (standard organic solvents are not listed in
the Material section). Prepare and store all reagents at room temperature (unless indicated otherwise).
2.1 Peptide
Synthesis
1. Silicycle MiniBlock® 10 mL polypropylene disposable reaction
tubes (SiliCycle Inc., Que ´bec, Canada) or equivalent.
2. Wang resins preloaded with the C-terminal amino acid or
2-chlorotrityl chloride resin.
3. 9-Fluorenyl-methyloxycarbonyl (Fmoc)-protected amino acids
with standard side-chain protection groups.
4. 0.5 M 2-(1-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) or 0.5 M 2-(1H-7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU) in dimethylformamide (DMF). Dissolve
1.9 g HBTU or 1.9 g HATU in 10 mL DMF and store
solutions in light-protected bottles.
5. 20% (v/v) piperidine in DMF.
6. Kaiser test kit (Sigma-Aldrich Corp, St. Louis, USA).
7. Capping solution: 10% (v/v) acetic anhydride, 10% (v/v) N,Ndiisopropylethylamine (DIPEA), 80% (v/v) DMF. Mix 1 mL of
acetic anhydride, 1 mL of DIPEA in 8 mL of DMF. Prepare
solution fresh before use.
8. Cleavage cocktail: 80% (v/v) trifluoroacetic acid (TFA), 10%
(v/v) water, 10% (v/v) Triisopropylsilane (TIPS).
9. Ice-cold diethyl ether: store at À20
C until usage.
10. Cy5-Maleimide (GE Healthcare, Chicago, USA).
11. Polyethylene glycol (PEG)-diacid (Iris Biotech GmbH, Marktredwitz, Germany).
12. Biotin (lyophilized powder).
13. PBS buffer: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 ,
2 mM KH 2 PO 4 , pH 7.4. Dissolve 8 g NaCl, 200 mg KCl,
1.44 g Na 2 HPO 4 , and 240 mg KH 2 PO 4 in 800 mL distilled
water. Adjust the pH to 7.4 by adding either 0.1 M NaOH or
0.1 M HCl. Fill up to a total volume of 1 L with distilled water.
14. Triphenylphosphine (PPh 3 ).
15. Diisopropyl azodicarboxylate (DIAD).
16. Diazabicyclo[5.4.0]undec-7-ene (DBU).
17. 2,4,6-trimethylpyridine (Collidine).
160
Dominik J. Essig et al.