title: Enzyme-linked immunosorbent assay for trkA tyrosine kinase activity.
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.
authors.affiliation: Department of Cell Biology, Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania, 19380, USA.
authors.forename: T S
authors.initials: TS
authors.lastname: Angeles
authors.forename: C
authors.initials: C
authors.lastname: Steffler
authors.forename: B A
authors.initials: BA
authors.lastname: Bartlett
authors.forename: R L
authors.initials: RL
authors.lastname: Hudkins
authors.forename: R M
authors.initials: RM
authors.lastname: Stephens
authors.forename: D R
authors.initials: DR
authors.lastname: Kaplan
authors.forename: C A
authors.initials: CA
authors.lastname: Dionne
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001597:Benzylidene Compounds / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology*; D002413:Cations, Divalent; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D026121:Indole Alkaloids; D007700:Kinetics; D009570:Nitriles / Q000494:pharmacology; D020917:Receptor, trkA / Q000032:analysis* / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins; D010738:Type C Phospholipases / Q000378:metabolism; D020032:Tyrphostins
chemical_list: C081020:AG-879; D001597:Benzylidene Compounds; D002227:Carbazoles; D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D026121:Indole Alkaloids; D009570:Nitriles; D011994:Recombinant Proteins; D020032:Tyrphostins; D000255:Adenosine Triphosphate; C049985:staurosporine aglycone; D020917:Receptor, trkA; D010738:Type C Phospholipases