High-sensitivity detection and quantitative analysis of native protein-protein interactions and multiprotein complexes by flow cytometry.
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.
Schrum
Adam G
AG
Department of Research, University Hospital-Basel, Basel, Switzerland. Schrum.Adam@mayo.edu
Gil
Diana
D
Dopfer
Elaine P
EP
Wiest
David L
DL
Turka
Laurence A
LA
Schamel
Wolfgang W A
WW
Palmer
Ed
E
D000818:Animals; D017252:CD3 Complex / Q000378:metabolism; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D047468:Immunoprecipitation; D051379:Mice; D008807:Mice, Inbred BALB C; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism
D017252:CD3 Complex; D046912:Multiprotein Complexes; D011506:Proteins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase