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Measuring Protein–Protein Interactions Using Biacore

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Protein Chromatography

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1485))

Abstract

The use of optical biosensors for studying macromolecular interactions is gaining increasing popularity. In one study, 1514 papers that involved the application of biosensor data were identified for the year 2009 alone (Rich and Myszka, J Mol Recognit 24:892–914, 2011), the sheer volume and variety of which present a daunting task for the burgeoning biosensor user to accumulate and decipher. This chapter is designed to provide the reader with the tools necessary to prepare, design, and efficiently execute a kinetic experiment on Biacore. It is written to guide the Biacore user through basic theory, system maintenance, and assay setup while also offering some practical tips that we find useful for Biacore-based studies. Many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To highlight these procedures, this protocol describes the kinetic characterization of single chain Fv (scFv) antibody fragments from crude bacterial lysates using an antibody affinity capture approach. Even though we specifically describe the capture of HA-tagged scFv antibody fragments to an anti-HA tag monoclonal antibody-immobilized surface prior to kinetic analysis, the same methodologies are universally applicable and can be used for practically any affinity pair and most Biacore systems.

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Author information

Authors and Affiliations

  1. Biomedical Diagnostics Institute, Dublin City University, Dublin 9, Ireland

    Paul Leonard, Stephen Hearty, Hui Ma & Richard O’Kennedy

  2. School of Biotechnology, Dublin City University, Dublin 9, Ireland

    Paul Leonard, Stephen Hearty & Richard O’Kennedy

  3. National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland

    Paul Leonard, Stephen Hearty & Richard O’Kennedy

Authors
  1. Paul Leonard
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  2. Stephen Hearty
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  3. Hui Ma
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  4. Richard O’Kennedy
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Corresponding author

Correspondence to Richard O’Kennedy .

Editor information

Editors and Affiliations

  1. School of Biotechnology, Dublin City University, Dublin, Ireland

    Dermot Walls

  2. Department of Applied Sciences, Dundalk Institute of Technology, Dublin, Ireland

    Sinéad T. Loughran

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Leonard, P., Hearty, S., Ma, H., O’Kennedy, R. (2017). Measuring Protein–Protein Interactions Using Biacore. In: Walls, D., Loughran, S. (eds) Protein Chromatography. Methods in Molecular Biology, vol 1485. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6412-3_17

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  • DOI: https://doi.org/10.1007/978-1-4939-6412-3_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6410-9

  • Online ISBN: 978-1-4939-6412-3

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