Abstract
Most proteins need to interact physically with other proteins to be able to perform their biological functions; the identification of new protein-binding partners for a protein of interest may therefore be a key strategy in defining the mechanism of action for a particular protein. Many different approaches to studying protein-protein interactions exist, but identifying which interactions are biologically significant is not trivial. Thus, once an interaction is found, the first step is to validate the suggested interactions using genetic, biochemical, and cell-biological approaches. Here we will focus on three complementary protein interaction approaches: (a) the yeast two-hybrid (YTH) system, a genetic assay performed in living yeast cells, which can be used both to identify proteins that bind to a protein of interest and to determine domains or residues critical for an interaction; and two biochemical approaches, (b) the coimmunoprecipitation of interacting proteins with a specific antibody; and (c) the far-Western method that can show direct physical interaction of two proteins.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Pedrazzi, G., Perrera, C., Blaser, H., et al. (2001) Direct association of Bloom’s syndrome gene product with the human mismatch repair protein MLH1. Nucleic Acids Res. 29, 4378–4386.
Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Bartel, P. L., Chien, C.-T., Sternglanz, R., and Fields, S. (1993) Using the two-hybrid system to detect protein-protein interactions, in: Cellular Interactions in Development: A Practical Approach (Hartley, D. A., ed.), Oxford University Press, Oxford, England, pp. 153–179.
Toby, G. G. and Golemis, E. A. (2001) Using the yeast interaction trap and other two-hybrid-based approaches to study protein-protein interactions. Methods. 24, 201–217.
Walhout, A. J., Boulton, S. J., and Vidal, M. (2000) Yeast two-hybrid systems and protein interaction mapping projects for yeast and worm. Yeast. 17, 88–94.
Durfee, T., Becherer, K., Chen, P.-L., et al. (1993) The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 7, 555–569.
Gavin, A. C., Bosche, M., Krause R., et al. (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature. 415, 141–147.
Ho, Y., Gruhler, A., Heilbut, A. et al. (2002) Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature. 415, 180–183.
Stagljar, I., Bourquin, J.-P., and Schaffner, W. (1996) Use of the two-hybrid system and random sonicated DNA to identify the interaction domain of a protein. BioTechniques. 21, 430–432.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Pedrazzi, G., Stagljar, I. (2004). Protein-Protein Interactions. In: Lieberman, H.B. (eds) Cell Cycle Checkpoint Control Protocols. Methods in Molecular Biology™, vol 241. Humana Press. https://doi.org/10.1385/1-59259-646-0:269
Download citation
DOI: https://doi.org/10.1385/1-59259-646-0:269
Publisher Name: Humana Press
Print ISBN: 978-1-58829-115-8
Online ISBN: 978-1-59259-646-1
eBook Packages: Springer Protocols