Peptide-Based Drug Design

64 Winkler and Campbell
3. For incubation of the membrane with the biotinylated protein sample and streptavidin,
follow the instructions of steps 1–4 in Subheading 3.3.1. with the exception
of using the HRP-labeled streptavidin at a concentration of 0.1 �g/mL instead of
the HRP-labeled proteins (antibodies).
4. Detection: Follow step 6 of Subheading 3.3.1. or step 2 of Subheading 3.3.2.
4. Notes
1. Reagents must be protected from moisture. To avoid condensation, reagent
bottles stored in the fridge or freezer should be unopened and warmed up to room
temperature for approx 30 min before use.
2. If the binding is expected to be very weak the use of PBS, TBS, PBS-T, or TBS-T
instead of blocking buffer is possible. In that case, due to possible unspecific
binding, a higher background signal during detection may occur.
3. To avoid problems due to formation of poor solubility in the urea, mix the in situ
activated solutions for about 30 min. Then centrifuge the mixtures and transfer
the supernatant to vials for the synthesis.
4. Because of the flexibility and linear structure of the molecule, �-alanine is
commonly used for modification of the cellulose as well as a spacer to achieve a
higher distance of the amino functions from the cellulose matrix. However, other
functionalizations are also possible (65).
5. The membrane can be stored at −80oC for several months until needed. For
longer storage a slight loss of loading is possible. Storage of the membranes at
−80◦C is recommended. The membranes can also be stored at −20◦Cor+4◦C, but there is a higher risk of losing activity over a long storage time.
6. Do not stain the spots too strongly because if the the amount of absorbed
bromophenol blue is too high, some of this dye could be incorporated in the
peptide. In that case removal of the color is almost impossible and could affect
the detection. Because bromophenol blue has a blue color in a basic environment,
e.g., primary amino functions, a difference in the intensity of the color of the
spots is normal due to different acidity of the coupled amino acids.
7. To cover the whole area of large spots, it is recommended to use at least 20%
more amino acid solution volume than in step 5 of Subheading 3.1.
8. Release of peptides with ammonia might work only on homemade membranes.
Commercially available membranes with amino functional groups often have
other than ester bonds between the spacer and cellulose. For such membranes
the use of additional linkers might be necessary (e.g., thioester (66), HMB linker
(67), Rink linker (68)).
9. To minimize possible contamination with side products from the edge of the spot,
the diameter of the punched-out discs should be smaller than the spot diameter.
10. In general, the peptide sequences should be between 6 and 15 amino acids in
length; spot syntheses of longer (e.g., 34 and 44 amino acids (69,70)) and shorter
(e.g., 2 amino acids (71)) peptides are also described. With shorter peptides, the
measurable activity might be too low. For the synthesis of long peptides, the