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
Part of the Neuromethods book series (NM, volume 146)


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.


Immunoprecipitation Blue native polyacrylamide gel electrophoresis Protein complexes Mass spectrometry Proteomics 


  1. 1.
    Li KW, Chen N, Klemmer P, Koopmans F, Karupothula R, Smit AB (2012) Identifying true protein complex constituents in interaction proteomics: the example of the DMXL2 protein complex. Proteomics 12:2428–2432CrossRefGoogle Scholar
  2. 2.
    Morris JH, Knudsen GM, Verschueren E, Johnson JR, Cimermancic P, Greninger AL, Pico AR (2014) Affinity purification-mass spectrometry and network analysis to understand protein-protein interactions. Nat Protoc 9:2539–2554CrossRefGoogle Scholar
  3. 3.
    Pandya NJ, Klaassen RV, van der Schors RC, Slotman JA, Houtsmuller A, Smit AB, Li KW (2016) Group 1 metabotropic glutamate receptors 1 and 5 form a protein complex in mouse hippocampus and cortex. Proteomics 16:2698–2705CrossRefGoogle Scholar
  4. 4.
    Chen N, Pandya NJ, Koopmans F, Castelo-Szekelv V, van der Schors RC, Smit AB, Li KW (2014) Interaction proteomics reveals brain region-specific AMPA receptor complexes. J Proteome Res 13:5695–5706CrossRefGoogle Scholar
  5. 5.
    Schwenk J, Harmel N, Brechet A, Zolles G, Berkefeld H, Muller CS, Bildl W, Baehrens D, Huber B, Kulik A, Klocker N, Schulte U, Fakler B (2012) High-resolution proteomics unravel architecture and molecular diversity of native AMPA receptor complexes. Neuron 74:621–633CrossRefGoogle Scholar
  6. 6.
    Brechet A, Buchert R, Schwenk J, Boudkkazi S, Zolles G, Siquier-Pernet K, Schaber I, Bildl W, Saadi A, Bole-Feysot C, Nitschke P, Reis A, Sticht H, Al-Sanna'a N, Rolfs A, Kulik A, Schulte U, Colleaux L, Abou Jamra R, Fakler B (2017) AMPA-receptor specific biogenesis complexes control synaptic transmission and intellectual ability. Nat Commun 8:15910CrossRefGoogle Scholar
  7. 7.
    Heusel M, Bludau I, Rosenberger G, Hafen R, Frank M, Banaei-Esfahani A, Collins B, Gstaiger M, Aebersold R (2018) Complex-centric proteome profiling by SEC-SWATH-MS. bioRxivGoogle Scholar
  8. 8.
    Kirkwood KJ, Ahmad Y, Larance M, Lamond AI (2013) Characterization of native protein complexes and protein isoform variation using size-fractionation-based quantitative proteomics. Mol Cell Proteomics 12:3851–3873CrossRefGoogle Scholar
  9. 9.
    Schagger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199:223–231CrossRefGoogle Scholar
  10. 10.
    Munawar N, Olivero G, Jerman E, Doyle B, Streubel G, Wynne K, Bracken A, Cagney G (2015) Native gel analysis of macromolecular protein complexes in cultured mammalian cells. Proteomics 15:3603–3612CrossRefGoogle Scholar
  11. 11.
    Pardo M, Bode D, Yu L, Choudhary JS (2017) Resolving affinity purified protein complexes by blue native PAGE and protein correlation profiling. J Vis ExpGoogle Scholar

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

Personalised recommendations