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    Development of a flow cytometric co-immunoprecipitation technique for the study of multiple protein-protein interactions and its application to T-cell receptor analysis.

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    Authors
    Bridgeman, John S
    Blaylock, Morgan
    Hawkins, Robert E
    Gilham, David E
    Affiliation
    Cell Therapy Group, Cancer Research UK Department of Medical Oncology, The School of Cancer and Imaging Sciences, The University of Manchester, Manchester Academic Health Science Centre, Manchester, United Kingdom.
    Issue Date
    2010-04
    
    Metadata
    Show full item record
    Abstract
    Co-immunoprecipitation is the classical approach for investigating protein-protein interactions. Analysis is generally conducted using the Western blot approach. We set out to investigate whether flow cytometry was a feasible alternative to Western blotting. Using the TCR-CD3 complex as a model for intermolecular interactions in the MA5.8 cell line, FLAG-tagged CD3zeta-scFv fusion proteins could be captured on anti-FLAG coupled beads and associated TCRbeta molecules could be detected by flow cytometry. This association was abrogated by mutations to the CD3zeta transmembrane domain. Using multicolor flow cytometry, TCRbeta, CD3epsilon, and the scFv region of the CD3zeta fusion molecule could all be detected from a single sample. This multicolor analysis was then applied to demonstrate the importance of correct lysis conditions for extraction of the TCR complex. In summary, this flow cytometric immunoprecipitation technique is a feasible alternative to classical co-immunoprecipitation analysis technique and offers many potential advantages including rapid analysis with increased target sensitivity, reduced technical demands, amenable to multiple protein analysis from a single sample, and provides a framework that may facilitate the development of high throughput analytical assays investigating protein-protein interactions.
    Citation
    Development of a flow cytometric co-immunoprecipitation technique for the study of multiple protein-protein interactions and its application to T-cell receptor analysis. 2010, 77 (4):338-46 Cytometry A
    Journal
    Cytometry. Part A
    URI
    http://hdl.handle.net/10541/109404
    DOI
    10.1002/cyto.a.20840
    PubMed ID
    20033991
    Type
    Article
    Language
    en
    ISSN
    1552-4930
    ae974a485f413a2113503eed53cd6c53
    10.1002/cyto.a.20840
    Scopus Count
    Collections
    All Paterson Institute for Cancer Research

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