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

✍ Scribed by Fridtjof Lund-Johansen; Ken Davis; James Bishop; Rene de Waal Malefyt

Publisher
John Wiley and Sons
Year
2000
Tongue
English
Weight
205 KB
Volume
39
Category
Article
ISSN
0196-4763

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✦ Synopsis

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 chain of the T-cell receptor, ZAP-70, CD3, CD5, SHP-1, and ERK-2, using 1-3 g 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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