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Enrichment of Low-Abundant Protein Targets by Immunoprecipitation Upstream of Mass Spectrometry

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Proteomic Profiling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1295))

Abstract

Immunoprecipitation (IP) is commonly used upstream of mass spectrometry (MS) as an enrichment tool for low-abundant protein targets. However, several aspects of the classical IP procedure such as nonspecific protein binding to the isolation matrix, detergents or high salt concentrations in wash and elution buffers, and antibody chain contamination in elution fractions render it incompatible with downstream mass spectrometry analysis. Here, we discuss two IP workflows that are designed to minimize or eliminate these contaminants: the first employs biotinylated antibodies and streptavidin magnetic beads while the second method utilizes a traditional antibody that is oriented and cross-linked to Protein AG magnetic beads. Both modified magnetic supports have low background binding and both antibody immobilization strategies significantly reduce or eliminate antibody heavy and light chain contamination in the eluent, minimizing potential ion suppression effects and thereby maximizing detection of target antigens and interacting proteins.

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Author information

Authors and Affiliations

  1. Research & Development, Thermo Fisher Scientific, 3747 N. Meridian Rd., Rockford, IL, 61101, USA

    Barbara Kaboord Ph.D., Suzanne Smith, Bhavin Patel & Scott Meier

Authors
  1. Barbara Kaboord Ph.D.
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  2. Suzanne Smith
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  3. Bhavin Patel
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  4. Scott Meier
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Corresponding author

Correspondence to Barbara Kaboord Ph.D. .

Editor information

Editors and Affiliations

  1. Bio-Rad Laboratories GmbH, Munich, Germany

    Anton Posch

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Kaboord, B., Smith, S., Patel, B., Meier, S. (2015). Enrichment of Low-Abundant Protein Targets by Immunoprecipitation Upstream of Mass Spectrometry. In: Posch, A. (eds) Proteomic Profiling. Methods in Molecular Biology, vol 1295. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2550-6_12

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  • DOI: https://doi.org/10.1007/978-1-4939-2550-6_12

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2549-0

  • Online ISBN: 978-1-4939-2550-6

  • eBook Packages: Springer Protocols

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