Development of a flow cytometric co-immunoprecipitation technique for the study of multiple protein-protein interactions and its application to T-cell receptor analysis.
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.
Bridgeman
John S
JS
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.
Blaylock
Morgan
M
Hawkins
Robert E
RE
Gilham
David E
DE
D017252:CD3 Complex / Q000378:metabolism; D002457:Cell Extracts; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003116:Color; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology; D047468:Immunoprecipitation / Q000379:methods*; D011485:Protein Binding; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014161:Transduction, Genetic
D017252:CD3 Complex; C121704:CD3 antigen, zeta chain; D002457:Cell Extracts; D007135:Immunoglobulin Variable Region; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins