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High-Throughput Quantification of SH2 Domain–Phosphopeptide Interactions with Cellulose–Peptide Conjugate Microarrays

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SH2 Domains

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

Abstract

The Src Homology 2 (SH2) domain family primarily recognizes phosphorylated tyrosine (pY) containing peptide motifs. The relative affinity preferences among competing SH2 domains for phosphopeptide ligands define “specificity space,” and underpins many functional pY mediated interactions within signaling networks. The degree of promiscuity exhibited and the dynamic range of affinities supported by individual domains or phosphopeptides is best resolved by a carefully executed and controlled quantitative high-throughput experiment. Here, I describe the fabrication and application of a cellulose–peptide conjugate microarray (CPCMA) platform to the quantitative analysis of SH2 domain specificity space. Included herein are instructions for optimal experimental design with special attention paid to common sources of systematic error, phosphopeptide SPOT synthesis, microarray fabrication, analyte titrations, data capture, and analysis.

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Author information

Authors and Affiliations

  1. Department of Human Genetics, The University of Chicago, Chicago, IL, USA

    Brett W. Engelmann

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  1. Brett W. Engelmann
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Correspondence to Brett W. Engelmann .

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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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Engelmann, B.W. (2017). High-Throughput Quantification of SH2 Domain–Phosphopeptide Interactions with Cellulose–Peptide Conjugate Microarrays. 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_21

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  • DOI: https://doi.org/10.1007/978-1-4939-6762-9_21

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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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