Enzyme-linked immunosorbent assay for trkA tyrosine kinase activity

Anal Biochem. 1996 Apr 5;236(1):49-55. doi: 10.1006/abio.1996.0130.

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.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Benzylidene Compounds / pharmacology
  • Carbazoles / pharmacology*
  • Cations, Divalent
  • Enzyme Inhibitors / pharmacology*
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Humans
  • Indole Alkaloids
  • Kinetics
  • Nitriles / pharmacology
  • Receptor, trkA / analysis*
  • Receptor, trkA / antagonists & inhibitors
  • Recombinant Proteins
  • Type C Phospholipases / metabolism
  • Tyrphostins*

Substances

  • AG-879
  • Benzylidene Compounds
  • Carbazoles
  • Cations, Divalent
  • Enzyme Inhibitors
  • Indole Alkaloids
  • Nitriles
  • Recombinant Proteins
  • Tyrphostins
  • Adenosine Triphosphate
  • staurosporine aglycone
  • Receptor, trkA
  • Type C Phospholipases