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Updated: Aug 11, 2026
IP-FCM: Immunoprecipitation Detected by Flow Cytometry
Published on: December 2, 2010
Flow cytometric analysis of immunoprecipitates: high-throughput analysis of protein phosphorylation and
F Lund-Johansen1, K Davis, J Bishop
1DNAX Research Institute for Cellular and Molecular Biology, Palo Alto, California, USA. f-johans@online.no
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.
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