A nonradioactive dot-blot assay for protein tyrosine kinase activity

A new procedure for the assay of protein tyrosine kinase, based on the detection of phosphorylated tyrosyl residues by using monoclonal antibodies to phosphotyrosine, is described. After incubation of a protein tyrosine kinase sample with the substrates poly-(GluNa,Tyr)4:1 and unlabeled ATP an aliquot of the reaction mixture is transferred to a polyvinylidene difluoride membrane. The extent of tyrosine phosphorylation is measured by probing the membrane with antiphosphotyrosine antibody followed by detection by the immunogold silver staining procedure. The signal is quantified by densitometry. The assay is linear with time and is quantitative in a wide range of sample protein concentrations. Its sensitivity allows the kinetic characterization of protein tyrosine kinases at low substrate concentrations, whereas on the other hand the avoidance of radioactivity enables the use of high ATP concentrations as well. Protein tyrosine kinase activities of human breast carcinomas and normal breast tissues measured with this method correlated well with the conventional assay, in which the incorporation of [32P]phosphate is measured by TCA precipitation and liquid scintillation counting. Compared to the latter, the new assay is at least as sensitive and accurate and harbors the advantage of the avoidance of radioactivity, thus enabling one to perform a large number of protein tyrosine kinase assays simultaneously.