Visualization of multiprotein complexes by flow cytometry.

Current Protocols in Immunology
Adam G Schrum

Abstract

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 co-associated molecules on the beads. The method represents a robust technique to assess native protein-protein interactions without requiring genetic engineering or large sample sizes.

References

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Jun 7, 2007·Science's STKE : Signal Transduction Knowledge Environment·Adam G SchrumEd Palmer

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Citations

Dec 24, 2010·Journal of Visualized Experiments : JoVE·Tessa R Davis, Adam G Schrum
Aug 4, 2016·Science Signaling·Stephen E P SmithAdam G Schrum
Jun 15, 2011·The Journal of Immunology : Official Journal of the American Association of Immunologists·Adam G SchrumEd Palmer
Oct 29, 2021·Frontiers in Immunology·Xiang ZhaoJoanna Brzostek

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