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

Abstract

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.

Approaching clinical proteomics: current state and future fields of application in fluid proteomics

“…Combining both forms of beads, one could scale up to many more parameters in one test (97). Multiplexed analysis of protein-protein interactions (111), or the simultaneous characterization of the binding of autoantibodies to multiple epitopes (112) are promising applications of flow cytometric bead-based assays.…”

Mutations in genes required for T-cell development:IL7R, CD45, IL2RG, JAK3, RAG1, RAG2, ARTEMIS, and ADA and severe combined immunodeficiency: HuGE review

“…The first type of biomarker is the family of cell membrane proteins unique to T cells, most notably CD3. Measurements of CD3 eluted from cultured T cells have shown strong correlation with T-cell count, 92 but this method has not yet been tested in whole blood or blood spots. Measurements of eluted CD4 also correlate strongly with T-cell counts, and this method has been applied successfully to whole blood and to DBS.…”

Ligand-Receptor-G-Protein Molecular Assemblies on Beads for Mechanistic Studies and Screening by Flow Cytometry

“…Specific subunit interactions could be assessed as a GFP-receptor binds to a subset of the combinations. Commercial hardware and software are already available for decoding the results of soluble, multiplex cytometric arrays (Lund-Johansen et al, 2000). Our standard coating of the nickel beads uses 0.7 pmol of G␣␤␥ per assay to obtain a 3:1 ratio of total signal to nonspecific signal, whereas the anti-FLAG beads use 0.17 pmol of G␣␤␥ per assay to obtain the same 3:1 ratio, using tens of thousands of beads.…”