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Enzyme-linked immunosorbent assay for trkA tyrosine kinase activity
T S Angeles1, C Steffler, B A Bartlett
1Department of Cell Biology, Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania, 19380, USA.
Abstract:
A 96-well microtiter enzyme-linked immunosorbent assay was developed to assay the activity of the cytoplasmic domain of trkA tyrosine kinase. The assay involves immobilization of phospholipase C- gamma/glutathione S-transferase fusion protein on a microtiter plate, addition of the kinase reaction mixture, and detection by an antibody to phosphotyrosine followed by an alkaline phosphatase-conjugated second antibody. The substrate used in this system, phospholipase C-gamma, is one of several biologically important substrates for the phosphorylation reaction of receptor-linked tyrosine kinases. The assay was then used to characterize kinase inhibitory activities of various small molecules including analogs of K-252a.