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Integrated Immunoprecipitation: Blue Native Gel Electrophoresis—Mass Spectrometry for the Identification of Protein Subcomplexes

  • Sophie J. F. van der Spek
  • August B. Smit
  • Nikhil J. Pandya
Protocol
Part of the Neuromethods book series (NM, volume 146)

Abstract

Proteins typically act as components of poly-protein complexes, which may vary in composition and function, and may depend on subcellular localization and cellular state. To understand their biology, there is a need to identify the constituents in these various protein complexes. Conventional immunoprecipitation combined with proteomics analysis is nowadays routinely used to identify multiple interactors of a bait protein; however, it is unable to distinguish protein subcomplexes from one another. Blue native polyacrylamide gel electrophoresis is appropriate to separate individual protein complexes. Here, we describe a protocol that combines the specificity of immunoprecipitation, the size separating capacity of Blue native polyacrylamide gel electrophoresis, and subsequent mass spectrometry to delineate the protein constituents in subcomplexes of a targeted protein.

Keywords

Immunoprecipitation Blue native polyacrylamide gel electrophoresis Protein complexes Mass spectrometry Proteomics 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Sophie J. F. van der Spek
    • 1
  • August B. Smit
    • 2
  • Nikhil J. Pandya
    • 1
    • 3
  1. 1.Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Amsterdam NeuroscienceVrije Universiteit AmsterdamAmsterdamThe Netherlands
  2. 2.Department of Molecular and Cellular Neurobiology, Faculty of Science, Center for Neurogenomics and Cognitive Research, Amsterdam NeuroscienceVrije Universiteit AmsterdamAmsterdamThe Netherlands
  3. 3.Roche Pharma Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center BaselF. Hoffmann-La Roche LtdBaselSwitzerland

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