Open AccessFlow cytometric analysis of immunoprecipitates: High‐throughput analysis of protein phosphorylation and protein–protein interactions
Author(s) -
LundJohansen Fridtjof,
Davis Ken,
Bishop James,
de Waal Malefyt Rene
Publication year - 2000
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/(sici)1097-0320(20000401)39:4<250::aid-cyto2>3.0.co;2-s
Subject(s) - phosphorylation , dephosphorylation , protein phosphorylation , protein kinase a , flow cytometry , microbiology and biotechnology , epitope , immunoprecipitation , biology , kinase , blot , signal transduction , monoclonal antibody , chemistry , antibody , biochemistry , phosphatase , gene , immunology
Background Activation‐induced protein phosphorylation can be studied by Western blotting, but this method is time consuming and depends on the use of radioactive probes for quantitation. We present a novel assay for the assessment of protein phosphorylation based on latex particles and flow cytometry. Methods This method employs monoclonal antibodies coupled to latex particles to immobilize protein kinase substrates. Their phosphorylation status is assessed by reactivity with phosphoepitope‐specific antibodies. The amount of immobilized protein on the particles was analyzed by direct or indirect immunofluorescence with antibodies to nonphosphorylated epitopes. Results The assay allowed measurement of phosphorylation of multiple protein kinase substrates in stimulated T cells, including the ζ chain of the T‐cell receptor, ZAP‐70, CD3, CD5, SHP‐1, and ERK‐2, using 1–3 μg of total cell protein per sample. The assay provided high resolution of kinetics of phosphorylation and dephosphorylation. Interactions of protein kinase substrates with associated signaling molecules were demonstrated. Conclusions The novel assay allows high‐throughput quantitative measurement of protein modifications during signal transduction. Cytometry 39:250–259, 2000 © 2000 Wiley‐Liss, Inc.