Summary
Far-western blotting is a convenient method to characterize protein–protein interactions, in which protein samples of interest are immobilized on a membrane and then probed with a nonantibody protein. In contrast to western blotting, which uses specific antibodies to detect target proteins, far-western blotting detects proteins on the basis of the presence or the absence of binding sites for the protein probe. When specific modular protein binding domains are used as probes, this approach allows characterization of protein–protein interactions involved in biological processes such as signal transduction, including interactions regulated by posttranslational modification. We here describe a rapid and simple protocol for far-western blotting, in which GST-tagged Src homology 2 (SH2) domains are used to probe cellular proteins in a phosphorylation-dependent manner.
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Acknowledgments
We are grateful to P. Nollau for his effort for the early development of the method, C. Thompson for technical assistance, K. Claffey for breast cancer samples, and A. Das for a bacterial culture system. This work was partially supported by grants from Breast Cancer Alliance and Connecticut Breast Health Initiative (to K.M.) and NIH grant CA107785 (to B.J.M.).
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Machida, K., Mayer, B.J. (2009). Detection of Protein-Protein Interactions by Far-Western Blotting. In: Kurien, B., Scofield, R. (eds) Protein Blotting and Detection. Methods in Molecular Biology, vol 536. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-542-8_34
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DOI: https://doi.org/10.1007/978-1-59745-542-8_34
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