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SH2 Domains pp 307–330Cite as

Binding Assays Using Recombinant SH2 Domains: Far-Western, Pull-Down, and Fluorescence Polarization

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1555))

Abstract

Recognition of phosphotyrosine-containing sequences by SH2 domains confers specificity in tyrosine kinase pathways. By assessing interactions between isolated SH2 domains and their binding proteins, it is possible to gain insight into otherwise inaccessible complex cellular systems. Far-Western, pull-down, and fluorescence polarization (FP) have been frequently used for characterization of phosphotyrosine signaling. Here, we outline standard protocols for these established assays using recombinant SH2 domain, emphasizing the importance of appropriate sample preparation and assay controls.

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Acknowledgments

We thank Joshua Jadwin for his assistance with editing this chapter, and Bruce Mayer for his continuous encouragement and support. This study was partly supported by grant CA1154966 from the National Institutes of Health and Quest for CURES (QFC) grant from the Leukemia and Lymphoma Society (to K.M.).

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Authors and Affiliations

  1. Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, 400 Farmington Avenue, Farmington, CT, 06030, USA

    Kazuya Machida

  2. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Bernard Liu

Authors
  1. Kazuya Machida
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  2. Bernard Liu
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Corresponding author

Correspondence to Kazuya Machida .

Editor information

Editors and Affiliations

  1. Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, Connecticut, USA

    Kazuya Machida

  2. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Bernard A. Liu

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Machida, K., Liu, B. (2017). Binding Assays Using Recombinant SH2 Domains: Far-Western, Pull-Down, and Fluorescence Polarization. In: Machida, K., Liu, B. (eds) SH2 Domains. Methods in Molecular Biology, vol 1555. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6762-9_17

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  • DOI: https://doi.org/10.1007/978-1-4939-6762-9_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6760-5

  • Online ISBN: 978-1-4939-6762-9

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