Peptide-Based Drug Design

54 Winkler and Campbell
Fmoc-�-alanine/DMSO solution to all spot positions (see Note 4). For manual
synthesis we recommend marking the spot positions with a pencil and to use a
multistep pipettor (see Fig. 2). Repeat the delivery after 20 min. After that allow
the reaction to occur once more for at least 20 min.
2. Blocking nonspot areas: Place the membrane face-down in an appropriate amount
of capping solution in the box. Ensure that there are no air bubbles under the
surface. Do not shake! Allow the reaction to occur for at least 5 min. Remove the
liquid. Add an appropriate amount of capping solution with 2% DIPEA. Place
the membrane face-up in this solution and allow it to react for 20 min.
3. Fmoc-deprotection: Wash four times with DMF for at least 30 s each. Treat twice
with 20% piperidine/DMF for 5 min each. Wash four times with DMF for at least
30 s each. Wash at least twice with methanol or ethanol.
4. Staining (optional) (25): Stain the membrane by shaking with staining solution
in a box. If the staining solution change its color to blue very rapidly, renew
the solution. Let the reaction occur for at least 15 s until the spots are blue (see
Note 6). Wash at least twice with methanol or ethanol until the wash solution
remains colorless. If faster drying is necessary, an additional washing step with
diethyl ether (twice) is possible.
5. Dry the membrane. The membrane is now ready for the first coupling cycle.
6. Coupling of activated amino acids: The stepwise buildup of peptides starts
from the C-terminus. Deliver the prepared corresponding activated amino acid
solutions to the corresponding positions on the membrane with the desired
volume. Repeat the spotting after 20 min (see Note 7).
7. Blocking unreacted free amino groups on peptide fragments: Add an appropriate
amount of capping solution to the box. Place the membrane face-down in this
Fig. 2. Dependency of spot size on the manually delivered volume of Fmoc-�-Ala-
OPfp/DMSO solution on Whatman 50 filter paper.