Abstract
Understanding protein–protein interactions is a key step in unravelling the roles proteins play in cellular function. The ability to analyse protein–protein interactions rapidly and economically is a powerful research tool. Using peptide SPOT arrays, peptides of known sequence can be synthesized directly in discrete spots on a cellulose membrane and assayed for an interaction with a protein of interest. Several hundred peptides can be synthesized on each cellulose membrane; therefore, this method is amenable to designing high-throughput peptide binding studies. SPOT arrays are particularly well suited for deducing peptidic binding motifs within proteins that are difficult to purify in sufficient quantities for traditional biochemical analyses, as well as for determining binding specificities and targets for proteins of undefined function. Peptide SPOT arrays have been used extensively to define protein–protein interaction surfaces. In this chapter, we will outline the steps involved in designing and probing a peptide SPOT array to identify peptide binding motifs for a protein of interest.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Pawson, T. (2007) Dynamic control of signaling by modular adaptor proteins Curr Opin Cell Biol 19, 112–6
Kanduc, D., Serpico, R., Lucchese, A., and Shoenfeld, Y. (2008) Correlating low-similarity peptide sequences and HIV B-cell epitopes Autoimmun Rev 7, 291–6
Frank, R. (1992) Spot-synthesis: An easy technique for the positionally addressable, parallel chemical synthesis on a membrane support Tetrahedron 48, 9217–32
Reineke, U., Ivascu, C., Schlief, M., Landgraf, C., Gereicke, S., Zahn, G., Herzel, H., Volkmer-Enger, R., and Schneider-Mergener, J. (2002) Identification of distinct antibody epitopes and mimitopes from a peptide array of 5520 randomly generated sequences J Immunol Methods 267, 37–51
Ogunjimi, A. A., Briant, D. J., Pece-Barbara, N., LeRoy, C., DiGuglielmo, G. M., Kavsak, P., Rasmussen, R. K., Seet, B. T., Sicheri, F., and Wrana, J. L. (2005) Regulation of Smurf2 ubiquitin ligase activity by anchoring the E2 to the HECT domain Mol Cell 19, 297–308
Lim, C. S., Seet, B. T., Ingham, R. J., Gish, G., Matskova, L., Winberg, G., Ernberg, I., and Pawson, T. (2007) The K15 protein of Kaposi’s sarcoma-associated herpesvirus recruits the endocytic regulator Intersectin 2 through a selective SH3 domain interaction. Biochemistry 46, 9874–85
Smith, M. J., Hardy, W. R., Murphy, J. M., Jones, N., and Pawson, T. (2006) Screening for PTB domain binding partners and ligand specificity using proteome-derived NPXY peptide arrays Mol Cell Biol 26, 8461–74
Leung, G. C., Murphy, J. M., Briant, D. J., and Sicheri, F. (2009) Characterization of kinase target phosphorylation consensus motifs using peptide SPOT arrays. Methods Mol Biol
Acknowledgments
This work was supported by grants from the National Cancer Institute of Canada and the Canadian Institutes of Health Research to F.S., and from the Terry Fox Foundation through an award from National Cancer Institute of Canada to GCL. JMM is a recipient of a CJ Martin (Biomedical) Fellowship from the National Health and Medical Research Council of Australia.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Briant, D.J., Murphy, J.M., Leung, G.C., Sicheri, F. (2009). Rapid Identification of Linear Protein Domain Binding Motifs Using Peptide SPOT Arrays. In: Cretich, M., Chiari, M. (eds) Peptide Microarrays. Methods in Molecular Biology™, vol 570. Humana Press. https://doi.org/10.1007/978-1-60327-394-7_6
Download citation
DOI: https://doi.org/10.1007/978-1-60327-394-7_6
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-393-0
Online ISBN: 978-1-60327-394-7
eBook Packages: Springer Protocols