Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Peptide arrays on cellulose support: SPOT synthesis, a time and cost efficient method for synthesis of large numbers of peptides in a parallel and addressable fashion


Peptide synthesis on cellulose using SPOT technology allows the parallel synthesis of large numbers of addressable peptides in small amounts. In addition, the cost per peptide is less than 1% of peptides synthesized conventionally on resin. The SPOT method follows standard fluorenyl-methoxy-carbonyl chemistry on conventional cellulose sheets, and can utilize more than 600 different building blocks. The procedure involves three phases: preparation of the cellulose membrane, stepwise coupling of the amino acids and cleavage of the side-chain protection groups. If necessary, peptides can be cleaved from the membrane for assays performed using soluble peptides. These features make this method an excellent tool for screening large numbers of peptides for many different purposes. Potential applications range from simple binding assays, to more sophisticated enzyme assays and studies with living microbes or cells. The time required to complete the protocol depends on the number and length of the peptides. For example, 400 9-mer peptides can be synthesized within 6 days.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Preparation of the cellulose membrane.
Figure 2: Amino acid coupling cycle.
Figure 3: Modifications and final deprotection of the peptide.
Figure 4: Dependency of the spot size from the pipetted volume of DMSO on different filter papers ( Whatman 540; ▪ Whatman 50; Whatman Chr).
Figure 5: Picture of a cellulose sheet where 160 peptides were synthesized.
Figure 6: List of 13 different peptide sequences.
Figure 7: A typical HPLC profile of a crude 9-mer peptide with the D-amino-acid sequence NH2–mdeaffd–A′-A′-CONH2 containing two β-Ala (A′) as a spacer.
Figure 8: A MALDI-ToF-MS image of a biotinylated and cystein-cyclized crude 10-mer peptide with the sequence biotin-CSHFNDYC-A′A′-CONH2 containing two β-Ala (A′) as a spacer (calc. MW = 1,353.47).


  1. Sewald, N. & Jakubke, H.-D. Peptides—Chemistry and Biology 1st edn. (Wiley-VCH, Weinheim, Germany, 2002).

    Book  Google Scholar 

  2. Paschke, M. Phage display systems and their applications. Appl. Microbiol. Biotechnol. 70, 2–11 (2006).

    Article  CAS  Google Scholar 

  3. Westerlund-Wikstrom, B. Peptide display on bacterial flagella: principles and applications. Int. J. Med. Microbiol. 290, 223–230 (2000).

    Article  CAS  Google Scholar 

  4. Yan, X. & Xu, Z. Ribosome-display technology: applications for directed evolution of functional proteins. Drug Discov. Today 11, 911–916 (2006).

    Article  CAS  Google Scholar 

  5. Merrifield, R.B. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85, 2149–2154 (1963).

    Article  CAS  Google Scholar 

  6. McKay, F.C. & Albertson, N.F. New amine-masking groups for peptide synthesis. J. Am. Chem. Soc. 79, 4686–4690 (1957).

    Article  CAS  Google Scholar 

  7. Carpino, L.A. Oxidative reaction of hydrazines. IV. Elimination of nitrogen from 1,1-disubstituted-2-arenesulfonhydrazides. J. Am. Chem. Soc. 79, 4427–4431 (1957).

    Article  CAS  Google Scholar 

  8. Anderson, G.W. & McGregor, A.C. t-Butyloxycarbonylamino acids and their use in peptide synthesis. J. Am. Chem. Soc. 79, 6180–6183 (1957).

    Article  CAS  Google Scholar 

  9. Carpino, L.A. & Han, G.Y. The 9-fluorenylmethoxycarbonyl amino-protecting group. J. Org. Chem. 37, 3404–3409 (1972).

    Article  CAS  Google Scholar 

  10. Houghten, R.A. General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen–antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 82, 5131–5135 (1985).

    Article  CAS  Google Scholar 

  11. Pellois, J.P. et al. Individually addressable parallel peptide synthesis on microchips. Nat. Biotechnol. 20, 922–926 (2002).

    Article  CAS  Google Scholar 

  12. Geysen, H.M., Meloen, R.H. & Barteling, S.J. Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA 81, 3998–4002 (1984).

    Article  CAS  Google Scholar 

  13. Frank, R. Spot synthesis: an easy technique for positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 48, 9217–9232 (1992).

    Article  CAS  Google Scholar 

  14. Kramer, A. & Schneider-Mergener, J. Synthesis and screening of peptide libraries on continuous cellulose membrane supports. Methods Mol. Biol. 87, 25–39 (1998).

    CAS  PubMed  Google Scholar 

  15. Toepert, F. et al. Combining SPOT synthesis and native peptide ligation to create large arrays of WW protein domains. Angew. Chem. Int. Ed. Engl. 42, 1136–1140 (2003).

    Article  CAS  Google Scholar 

  16. Heine, N. et al. Synthesis and screening of peptoid arrays on cellulose membranes. Tetrahedron 59, 9919–9930 (2003).

    Article  CAS  Google Scholar 

  17. Blackwell, H.E. Hitting the SPOT: small-molecule macroarrays advance combinatorial synthesis. Curr. Opin. Chem. Biol. 10, 203–212 (2006).

    Article  CAS  Google Scholar 

  18. Hilpert, K., Hansen, G., Wessner, H., Volkmer-Engert, R. & Hohne, W. Complete substitutional analysis of a sunflower trypsin inhibitor with different serine proteases. J. Biochem. (Tokyo) 138, 383–390 (2005).

    Article  CAS  Google Scholar 

  19. Kramer, A. et al. Molecular basis for the binding promiscuity of an anti-p24 (HIV-1) monoclonal antibody. Cell 91, 799–809 (1997).

    Article  CAS  Google Scholar 

  20. Kramer, A. et al. Spot synthesis: observations and optimizations. J. Pept. Res. 54, 319–327 (1999).

    Article  CAS  Google Scholar 

  21. Weiser, A.A. et al. SPOT synthesis: reliability of array-based measurement of peptide binding affinity. Anal. Biochem. 342, 300–311 (2005).

    Article  CAS  Google Scholar 

  22. Molina, F., Laune, D., Gougat, C., Pau, B. & Granier, C. Improved performances of spot multiple peptide synthesis. Pept. Res. 9, 151–155 (1996).

    CAS  PubMed  Google Scholar 

  23. Geysen, H.M., Barteling, S.J. & Meloen, R.H. Small peptides induce antibodies with a sequence and structural requirement for binding antigen comparable to antibodies raised against the native protein. Proc. Natl. Acad. Sci. USA 82, 178–182 (1985).

    Article  CAS  Google Scholar 

  24. Houghten, R.A. et al. Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery. Nature 354, 84–86 (1991).

    Article  CAS  Google Scholar 

  25. Zander, N., Beutling, U., Dikmans, A., Thiele, S. & Frank, R. A special cellulose membrane support for the combinatorial and parallel synthesis of peptide libraries suitable for the SC2-type manufacturing of high density multi-purpose chemical micro-arrays. in Peptides 2004: Proceedings of the Third International and 28th European Peptide Symposium (eds. Flegel, M. et al.) 405–406 (2005).

    Google Scholar 

  26. Hilpert, K., Hansen, G., Wessner, H., Schneider-Mergener, J. & Hohne, W. Characterizing and optimizing protease/peptide inhibitor interactions, a new application for spot synthesis. J. Biochem. (Tokyo) 128, 1051–1057 (2000).

    Article  CAS  Google Scholar 

  27. Hilpert, K., Volkmer-Engert, R., Walter, T. & Hancock, R.E. High-throughput generation of small antibacterial peptides with improved activity. Nat. Biotechnol. 23, 1008–1012 (2005).

    Article  CAS  Google Scholar 

  28. Hilpert, K. & Hancock, R.E.W. Use of luminescent bacteria for rapid screening and characterization of short cationic antimicrobial peptides synthesized on cellulose by peptide array technology. Nat Protocols (in the press).

  29. Kamradt, T. & Volkmer-Engert, R. Cross-reactivity of T lymphocytes in infection and autoimmunity. Mol. Divers. 8, 271–280 (2004).

    Article  CAS  Google Scholar 

  30. Reineke, U. et al. Mapping protein–protein contact sites using cellulose-bound peptide scans. Mol. Divers. 1, 141–148 (1996).

    Article  CAS  Google Scholar 

  31. Hilpert, K., Winkler, D.F.H. & Hancock, R.E.W. Cellulose-bound peptide arrays: preparation and applications. Biotech. Genet. Eng. Rev. 24 (in the press).

  32. Fields, G.B. & Noble, R.L. Solid phase synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Prot. Res. 35, 161–214 (1990).

    Article  CAS  Google Scholar 

  33. Krchnak, V., Vagner, J. & Lebl, M. Noninvasive continuous monitoring of solid-phase peptide synthesis by acid-base indicator. Int. J. Pept. Protein Res. 32, 415–416 (1988).

    Article  CAS  Google Scholar 

  34. Gausepohl, H. & Behn, C. Automated synthesis of solid-phase bound peptides. in Peptide Arrays on Membrane Support (eds. Koch, J. & Mahler, M.) 55–68 (Springer, Berlin, Heidelberg, 2002).

    Chapter  Google Scholar 

  35. Bowman, M.D., Jacobson, M.M., Pujanauski, B.G. & Blackwell, H.E. Efficient synthesis of small molecule macroarrays: optimization of the macroarray synthesis platform and examination of microwave and conventional heating methods. Tetrahedron 62, 4715–4727 (2006).

    Article  CAS  Google Scholar 

  36. Takahashi, M., Ueno, A. & Mihara, H. Peptide design based on an antibody complementarity-determining region (CDR): construction of porphyrin-binding peptides and their affinity maturation by a combinatorial method. Chem. Eur. J. 6, 3196–3203 (2000).

    Article  CAS  Google Scholar 

Download references


We are grateful for financial assistance from the Applied Food and Materials Network and the Canadian Institutes of Health Research. R.E.W.H. was supported by a Canada Research Chair award. K.H. was supported by a fellowship from the Canadian Institutes of Health Research. We thank R.A. Klady for the critical proofreading of the manuscript.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Kai Hilpert.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hilpert, K., Winkler, D. & Hancock, R. Peptide arrays on cellulose support: SPOT synthesis, a time and cost efficient method for synthesis of large numbers of peptides in a parallel and addressable fashion. Nat Protoc 2, 1333–1349 (2007).

Download citation

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing