Visualization of Multiprotein Complexes by Flow Cytometry

Adam G. Schrum1

1 Mayo Clinic College of Medicine, Rochester, Minnesota
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 5.9
DOI:  10.1002/0471142735.im0509s87
Online Posting Date:  November, 2009
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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.

Keywords: immunoprecipitation; flow cytometry; protein‐protein interactions; multiprotein complex

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Table of Contents

  • Introduction
  • Basic Protocol 1: Covalent Coupling of Antibody to CML Beads
  • Basic Protocol 2: Immunoprecipitation from Cell Lysates Analyzed by Flow Cytometry (IP‐FCM)
  • Support Protocol 1: Quantitative Flow Cytometry (qFCM)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Covalent Coupling of Antibody to CML Beads

  • CML (carboxylate‐modified latex) beads (∼5 µm surfactant‐free white carboxylate‐modified polystyrene latex beads; Interfacial Dynamics Corporation, cat. no. 2‐5000 or 2‐6000;; store up to 3 years (or longer) at 4°C
  • MES coupling buffer (see recipe)
  • EDAC‐MES solution (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.2 mg/ml Ab for immunoprecipitation (IP) in PBS (see recipe)
  • QBS buffer (see recipe)
  • Hemacytometer (Neubauer chamber) for bead counting (also see appendix 3A)
  • Microscope capable of 100× magnification for bead counting
  • Vibrating shaker, or Thermomixer (Eppendorf product 5436)
  • Additional reagents and equipment for use of a hemacytometer or Coulter Counter ( appendix 3A)

Basic Protocol 2: Immunoprecipitation from Cell Lysates Analyzed by Flow Cytometry (IP‐FCM)

  • Cells for lysis: primary lymphocytes
  • Lysis buffer (see recipe) with freshly added protease and phosphatase inhibitors (see reciperecipes), ice cold
  • IP beads ( protocol 1)
  • Post‐IP wash buffer (see recipe)
  • FCM staining buffer (see recipe)
  • Antibody probe(s), fluorochrome‐conjugated (≥0.2 mg/ml or as supplied by manufacturer)
  • End‐over‐end rotator (vertical)
  • 96‐well polypropylene plates with 0.2‐ml tall‐chimney‐bottom wells (Fisher, cat. no. 08‐408‐230)
  • Centrifuge with microtiter plate adapter
  • 5‐ml FACS tubes
  • Flow cytometer (also see Chapter 5)
  • Flow cytometry acquisition and analysis software, such as CellQuest (BD) and FlowJo (Treestar)
  • Additional reagents and equipment for flow cytometry (Chapter 5)

Support Protocol 1: Quantitative Flow Cytometry (qFCM)

  • Rainbow calibration particles (Spherotech product RCP‐30‐5A)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Flow cytometer
  • Flow cytometry acquisition and analysis software, such as CellQuest (BD) and FlowJo (Treestar)
  • Spreadsheet software (Microsoft Excel)
  • Curve generation and analysis software (such as Prism GraphPad)
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Literature Cited

   Conrotto, P., Yakymovych, I., Yakymovych, M., and Souchelnytskyi, S. 2007. Interactome of transforming growth factor‐beta type I receptor (TbetaRI): Inhibition of TGFbeta signaling by Epac1. J. Proteome Res. 6:287‐297.
   Ghavidel, A., Cagney, G., and Emili, A. 2005. A skeleton of the human protein interactome. Cell 122:830‐832.
   Gil, D., Schrum, A.G., Alarcon, B., and Palmer, E. 2005. T cell receptor engagement by peptide‐MHC ligands induces a conformational change in the CD3 complex of thymocytes. J. Exp. Med. 201:517‐522.
   Jares‐Erijman, E.A. and Jovin, T.M. 2006. Imaging molecular interactions in living cells by FRET microscopy. Curr. Opin. Chem. Biol. 10:409‐416.
   Lund‐Johansen, F., Davis, K., Bishop, J., and de Waal Malefyt, R. 2000. Flow cytometric analysis of immunoprecipitates: High‐throughput analysis of protein phosphorylation and protein‐protein interactions. Cytometry 39:250‐259.
   Parrish, J.R., Gulyas, K.D., and Finley, R.L. Jr. 2006. Yeast two‐hybrid contributions to interactome mapping. Curr. Opin. Biotechnol. 17:387‐393.
   Phizicky, E.M. and Fields, S. 1995. Protein‐protein interactions: Methods for detection and analysis. Microbiol. Rev. 59:94‐123.
   Schrum, A.G., Gil, D., Dopfer, E.P., Wiest, D.L., Turka, L.A., Schamel, W.W., and Palmer, E. 2007. High‐sensitivity detection and quantitative analysis of native protein‐protein interactions and multiprotein complexes by flow cytometry. Sci. STKE Jun 5; 2007(389):pl2.
   Smith, D.B. and Johnson, K.S. 1988. Single‐step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S‐transferase. Gene 67:31‐40.
   Stelzl, U. and Wanker, E.E. 2006. The value of high quality protein‐protein interaction networks for systems biology. Curr. Opin. Chem. Biol. 10:551‐558.
   Swamy, M., Siegers, G.M., Minguet, S., Wollscheid, B., and Schamel, W.W. 2006. Blue native polyacrylamide gel electrophoresis (BN‐PAGE) for the identification and analysis of multiprotein complexes. Sci. STKE July 25; 2006(345):pl4.
   Teixeiro, E., Daniels, M.A., Hausmann, B., Schrum, A.G., Naeher, D., Luescher, I., Thome, M., Bragado, R., and Palmer, E. 2004. T cell division and death are segregated by mutation of TCRbeta chain constant domains. Immunity 21:515‐526.
   Treves, S., Franzini‐Armstrong, C., Moccagatta, L., Arnoult, C., Grasso, C., Schrum, A., Ducreux, S., Zhu, M.X., Mikoshiba, K., Girard, T., Smida‐Rezgui, S., Ronjat, M., and Zorzato, F. 2004. Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion. J. Cell. Biol. 166:537‐548.
   Tu, L.C., Yan, X., Hood, L., and Lin, B. 2007. Proteomics analysis of the interactome of N‐myc downstream regulated gene 1 and its interactions with the androgen response program in prostate cancer cells. Mol. Cell Proteomics 6:575‐588.
   Tyers, M. and Mann, M. 2003. From genomics to proteomics. Nature 422:193‐197.
Key References
   Lund‐Johansen et al., 2000. See above.
  The first report of IP‐FCM.
   Schrum et al., 2007. See above.
  Describes the combination of IP‐FCM with quantitative flow cytometry for (1) analysis of stoichiometric ratios within multiprotein complexes, and (2) co‐IP data from rare primary cell subsets.
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