UNIT 5.9 Visualization of Multiprotein Complexes by Flow Cytometry
Published Online: 1 NOV 2009
Copyright © 2009 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Immunology
How to Cite
Schrum, A. G. 2009. Visualization of Multiprotein Complexes by Flow Cytometry. Current Protocols in Immunology. 87:5.9:5.9.1–5.9.14.
- Published Online: 1 NOV 2009
- Published Print: NOV 2009
Multiprotein complexes and other protein-protein interactions play important roles in virtually all cellular processes. Analysis of coimmunoprecipitation of protein complexes by flow cytometry (IP-FCM, or “the fly-p” method) provides a sensitive means to measure these interactions in the native/nondenatured state. First, immunoprecipitating antibodies are covalently coupled to polystyrene latex beads whose low autofluorescence is compatible with flow cytometry. These antibody-coupled beads are used to immunoprecipitate a specific protein (primary analyte) present in cell lysates. Finally, the protein complexes associated with the beads are probed with fluorochrome-conjugated antibodies specific for interaction partners, or secondary analytes, that may be associated with the primary analyte. The use of quantitative flow cytometric methodology can allow the semiquantitative fluorescence data generated to be converted into estimated numbers of coassociated molecules on the beads. The method represents a robust technique to assess native protein-protein interactions without requiring genetic engineering or large sample sizes. Curr. Protoc. Immunol. 87:5.9.1-5.9.14. © 2009 by John Wiley & Sons, Inc.
- flow cytometry;
- protein-protein interactions;
- multiprotein complex