Multiplex IP-FCM (immunoprecipitation-flow cytometry): Principles and guidelines for assessing physiologic protein-protein interactions in multiprotein complexes

Anya T. Bida, Diana Gil, Adam G. Schrum

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

There is significant interest in the development of methods with the potential to increase access to 'the interactome' for both experimental and clinical applications. Immunoprecipitation detected by flow cytometry (IP-FCM) is a robust, biochemical method that can be used for measuring physiologic protein-protein interactions (PPI) in multiprotein complexes (MPC) with high sensitivity. Because it is based on antibody-mediated capture of protein complexes onto microspheres, IP-FCM is potentially compatible with a multiplex platform that could allow simultaneous assessment of many physiologic PPI. Here, we consider the principles of ambient analyte conditions (AAC) and inter-bead independence, and provide a template set of experiments showing how to convert singleplex IP-FCM to multiplex IP-FCM, including assays to confirm the validity of the experimental conditions for data acquisition. We conclude that singleplex IP-FCM can be successfully upgraded to multiplex format, and propose that the unique strengths of multiplex IP-FCM make it a method that is likely to facilitate the acquisition of new PPI data from primary cell sources.

Original languageEnglish (US)
Pages (from-to)154-160
Number of pages7
JournalMethods
Volume56
Issue number2
DOIs
StatePublished - Feb 2012

Keywords

  • Flow cytometry
  • IP-FCM
  • Multiplex
  • Multiprotein complex
  • Protein-protein interaction

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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