Peptide-Based Drug Design

The Spot Technique 65
following rule must be kept in mind: the longer the peptide, the more difficulties
arise during coupling—the number of possible side reactions increases and
peptide purity decreases dramatically!
11. Since it is unlikely for one to be able to synthesize all possible sequences on
a single spot, do not start synthesis of a combinatorial library with more than
6 mixture positions. If necessary, start to screen 8mer peptides (6 mixture + 2
deconvoluted positions) and extend the peptide length with each synthesis round
by extending the deconvoluted positions and maintaining the number of mixed
positions (e.g., 6 mixture + 4 deconvoluted positions).
12. Because of possible strong side reactions during the probing, avoid the use
of cysteines in the first synthesis and screening rounds at defined amino acid
positions.
13. After incubation, if a spot signal also shows a corresponding spot signal by
reflection on a virtual diagonal (Pos.1,1 to pos. x,x), it is possible that these two
signals are caused by unspecific binding.
14. Avoid exposure to daylight at all steps using the chemiluminescence mixture. In
particular, when using X-ray film for detection a red light room should be used.
15. Use of towels made of nonrecycled paper is recommended. Using recycled towels
could lead to disturbances in detection by reactions with trace materials in the
paper.
16. After probing, the membrane must remain wet for regeneration. A previously
dried membrane is more difficult to regenerate than a wet one.
17. From time to time incomplete regeneration could occur. Therefore, it is recommended
to always use new membranes; if not, then the membrane should be
probed first with the control and then with the protein sample. For further probing
with the same membrane, completeness of the regeneration should be checked by
repeating the detection without the protein sample. All detectable signals should
be found in the previous control. Additional signals seen with the sample probe
should have disappeared. Stained spots are generally very hard to regenerate.
18. Commonly used biotinylating reagents are usually amine reactive. For biotinylation
of your protein, do not dissolve your protein sample in amine-containing
buffers, such as TBS, TBS-T, or PBS-T.
References
1. Frank, R. (1992) Spot-synthesis: An easy technique for the positionally addressable,
parallel chemical synthesis on a membrane support. Tetrahedron 48, 9217–9232.
2. Kramer, A. and Schneider-Mergener, J. (1998) Synthesis and application of peptide
libraries bound to continuous cellulose membranes. Methods Mol. Biol. 87, 25–39.
3. Gausepohl, H. and Behn, C. (2002) Automated synthesis of solid-phase bound
peptides. In: Peptide Arrays on Membrane Support, eds. J. Koch and M. Mahler,
pp. 55–68. Berlin: Springer-Verlag.
4. Molina, F., Laune, D., Gougat, C., Pau, B., and Granier, C. (1996) Improved performances
of spot multiple peptide synthesis. Peptide Res. 9, 151–155.