IMMUNOFLUORESCENT QUANTIFICATION OF TYROSINE-PHOSPHORYLATED PROTEINS BY FLOW CYTOMETRIC ANALYSIS

To measure the quantity of tyrosine phosphorylated proteins in cells, we have used a flow cytometry technique with fluorescein isothiocyanat e-labeled anti-phosphotyrosine antibody (FITC-alpha PY mAb). The analy sis was applied to the phosphotyrosine titration and showed an optimum amount of FITC-alpha PY mAb (30 mu g/l x 10(6)cells). The staining sp ecificity of our assay was tested by the addition of exogenous competi tors, showing a specific inhibition by phosphotyrosine but not by phos phoserine, phosphothreonine, or tyrosine. The assay was also able to e lucidate the inhibitory effect on tyrosine phosphorylation of genistei n, a protein tyrosine kinase inhibitor. These results imply that immun ofluorescent quantification assay using a flow cytometer could be a us eful technique to determine the intracellular level of tyrosine phosph orylated protein.