Flow cytometric analysis of immunoprecipitates: high-throughput analysis of protein phosphorylation and protein-protein interactions.
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 zeta chain of the T-cell receptor, ZAP-70, CD3, CD5, SHP-1, and ERK-2, using 1-3 microg 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.
Lund-Johansen
F
F
DNAX Research Institute for Cellular and Molecular Biology, Palo Alto, California, USA. f-johans@online.no
Davis
K
K
Bishop
J
J
de Waal Malefyt
R
R
D000818:Animals; D015153:Blotting, Western; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008863:Microspheres; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D019904:Polymethyl Methacrylate; D011233:Precipitin Tests / Q000379:methods*; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011817:Rabbits; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase
D008565:Membrane Proteins; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; C054979:antigen T cell receptor, zeta chain; D014443:Tyrosine; D019904:Polymethyl Methacrylate; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; C497262:Zap70 protein, mouse; D019950:Mitogen-Activated Protein Kinase 1