High-Sensitivity Detection and Quantitative Analysis of Native Protein-Protein Interactions and Multiprotein Complexes by Flow Cytometry
Adam G. Schrum1,2*,
Diana Gil3,
Elaine P. Dopfer4,
David L. Wiest5,
Laurence A. Turka6,
Wolfgang W. A. Schamel4, and
Ed Palmer1
1Department of Research, University Hospital-Basel, CH-4031 Basel, Switzerland.
2Department of Immunology, Mayo Clinic College of Medicine, 200 1st Street SW, Guggenheim 321a, Rochester, MN 55905, USA.
3Inmunología, Departamento de Microbiología I, Facultad de Medicina, Universidad Complutense de Madrid, Madrid 28043, Spain.
4Max-Planck-Institut für Immunbiologie und Universität Freiburg, Biologie III, Stübeweg 51, D-79108 Freiburg, Germany.
5Division of Basic Sciences, Immunobiology Working Group, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
6Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Abstract:
Most mechanisms of cell development, physiology, and signal transduction are controlled by protein-protein interactions. Immunoprecipitation of multiprotein complexes detected by flow cytometry (IP-FCM) is a means to quantitatively measure these interactions. The high sensitivity of this method makes it useful even when very little biomaterial is available for analysis, as in the case of rare primary cell subsets or patient samples. Detection of the T cell antigen receptor associated with the CD3 multiprotein complex from as few as 300 primary murine T cells is presented as an example. The method is compatible with quantitative flow cytometry techniques, making it possible to estimate the number of coimmunoprecipitated molecules. Both constitutive and inducible protein-protein interactions can be analyzed, as illustrated in related methodology using glutathione S-transferase–fusion protein pull-down experiments. IP-FCM represents a robust, quantitative, biochemical technique to assess native protein-protein interactions, without requiring genetic engineering or large sample sizes.
*Corresponding author. Telephone: (507) 255-2919; fax: (507) 284-1637; e-mail: Schrum.Adam{at}mayo.edu
