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

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 CD3f-scFv fusion proteins could be captured on anti-FLAG coupled beads and associated TCRb molecules could be detected by flow cytometry. This association was abrogated by mutations to the CD3f transmembrane domain. Using multicolor flow cytometry, TCRb, CD3e, and the scFv region of the CD3f 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. ' 2009 International Society for Advancement of Cytometry Key termsT-cell receptor; CD3f; co-immunoprecipitation; protein-protein interactions THE co-immunoprecipitation/Western blotting (co-ip/WB) approach has been the gold-standard technique for the investigation of suspected protein-protein interactions for many years. However, the co-ip/WB approach has limitations. For example, heavy and light chains from the capture antibodies can obscure identification of immuno-reactive bands on immuno-blots of the same molecular weight as the protein of interest. Furthermore, SDS-PAGE/WB can be a time-consuming process, particularly if multiple protein interactions are being investigated. To circumvent some of the issues surrounding standard co-ip/WB, we investigated the use of flow cytometry to analyze immunoprecipitates from a well-documented protein complex. The advantage of this approach being that the high throughput approach of flow cytometry would expedite the identification process and, with the large number of fluorochrome conjugated antibodies now available to the researcher, would permit analysis of multiple protein-protein interactions. Bead-based assays have steadily become adapted for flow cytometric purposes and can now be used to analyze kinase inhibitors (1), to quantify antibody binding sites (2), to detect low concentrations of DNA (3), and to measure cytokine secretion (4). Here, we show that a bead-based flow cytometric immunoprecipitation assay (FCIP) can be used to successfully analyze interactions between CD3f and the T-cell receptor (TCR) complex. Not only is

The Optimal Antigen Response of Chimeric Antigen Receptors Harboring the CD3ζ Transmembrane Domain Is Dependent upon Incorporation of the Receptor into the Endogenous TCR/CD3 Complex

“…We have recently reported the development of this technique (25). Using this approach, we have confirmed observations seen in MA5.8 cells.…”

“…Standard techniques to analyze the CAR-TCR interaction proved unsuccessful, so this led to the development of a novel bead-based flow technique (FCIP) that we have recently reported on (25). A C-terminal FLAG tag was cloned onto the CAR, and Jurkat T cells were transduced and sorted.…”

KCa1.1 channels regulate β1‐integrin function and cell adhesion in rheumatoid arthritis fibroblast‐like synoviocytes

“…Our studies utilized the technique of co-IP by flow cytometry, validated in a variety of systems (22)(23)(24)(25)(74)(75)(76)(77), to determine physical interactions between proteins in FLSs. This widely used technique has been compared to the traditional co-IP detected via Western blot analysis; co-IP by flow cytometry is as sensitive and qualitative but requires fewer cells per sample.…”

Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks

“…Using multi-colour IP-FCM, we reconstructed with high quantitative accuracy the dynamics of phosphorylations at the TCR-CD3 and ZAP70, which have previously been partially characterized by IP-WB and one-colour IP-FCM [7], [41]. These data have allowed us to develop a mechanistic model of the underlying TCR-CD3-ZAP70 interaction and reversible phosphorylations and (unlike the IP-WB data with their large error bars) have forced the model to precisely reproduce the kinetics of ZAP70 recruitment and phosphorylation.…”