Enzyme-linked immunosorbent assay for trkA tyrosine kinase activity.
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.
Angeles
T S
TS
Department of Cell Biology, Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania, 19380, USA.
Steffler
C
C
Bartlett
B A
BA
Hudkins
R L
RL
Stephens
R M
RM
Kaplan
D R
DR
Dionne
C A
CA
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
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