title: Multiplex IP-FCM (immunoprecipitation-flow cytometry): Principles and guidelines for assessing physiologic protein-protein interactions in multiprotein complexes.
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.
authors.affiliation: Mayo Clinic College of Medicine, Department of Immunology, Rochester, MN, USA.
authors.forename: Anya T
authors.initials: AT
authors.lastname: Bida
authors.forename: Diana
authors.initials: D
authors.lastname: Gil
authors.forename: Adam G
authors.initials: AG
authors.lastname: Schrum
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000918:Antibody Specificity; D045744:Cell Line, Tumor; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D005456:Fluorescent Dyes / Q000737:chemistry; D047468:Immunoprecipitation / Q000379:methods* / Q000592:standards; D051379:Mice; D008863:Microspheres; D046912:Multiprotein Complexes / Q000032:analysis* / Q000737:chemistry / Q000502:physiology; D025941:Protein Interaction Mapping / Q000379:methods* / Q000592:standards; D055550:Protein Stability; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D017186:Titrimetry
chemical_list: D000906:Antibodies; D005456:Fluorescent Dyes; D046912:Multiprotein Complexes; D011948:Receptors, Antigen, T-Cell