Flow cytometric analysis of immunoprecipitates: high-throughput analysis of protein phosphorylation and protein-protein interactions

Cytometry. 2000 Apr 1;39(4):250-9. doi: 10.1002/(sici)1097-0320(20000401)39:4<250::aid-cyto2>3.0.co;2-s.


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.

MeSH terms

  • Animals
  • Blotting, Western
  • Flow Cytometry / methods*
  • Humans
  • Lymphocyte Activation
  • Membrane Proteins / metabolism
  • Mice
  • Microspheres
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Polymethyl Methacrylate
  • Precipitin Tests / methods*
  • Protein Binding
  • Protein-Tyrosine Kinases / metabolism
  • Rabbits
  • Receptors, Antigen, T-Cell / metabolism
  • Substrate Specificity
  • T-Lymphocytes / chemistry
  • T-Lymphocytes / metabolism
  • Tyrosine / metabolism
  • ZAP-70 Protein-Tyrosine Kinase


  • Membrane Proteins
  • Phosphoproteins
  • Receptors, Antigen, T-Cell
  • antigen T cell receptor, zeta chain
  • Tyrosine
  • Polymethyl Methacrylate
  • Protein-Tyrosine Kinases
  • ZAP-70 Protein-Tyrosine Kinase
  • ZAP70 protein, human
  • Zap70 protein, mouse
  • Mitogen-Activated Protein Kinase 1