Kinase assay based on thiophosphorylation and biotinylation.
Protein kinases catalyze the transfer of the gamma-phosphate group from ATP to a serine, threonine or tyrosine residue of an acceptor protein. These enzymes play an important role in signal transduction. New inhibitors for these enzymes are actively being sought. In this article, we present a novel approach for detecting the activity of protein kinases, which could be useful for the high-throughput screening of chemical libraries. The method is based on the use of ATP gamma S instead of ATP in the phosphorylation reaction. This results in the transfer of a thiophosphate group onto a fluorescein-labeled acceptor peptide substrate. The mixture is then treated with a sulfur-reactive iodoacetyl derivative of biotin, which leads to the modification of the nucleophilic sulfur of the thiophosphate group and the generation of a fluorescently labeled, biotinylated molecule. Finally, streptavidin is added to the mixture and it binds to all biotinylated molecules present. The binding of streptavidin to the thiophosphorylated and biotinylated kinase substrate can be conveniently detected by measuring the change in fluorescence polarization of the fluorescent dye attached to the peptide. The detection of kinase inhibitors is demonstrated. The method is completely homogeneous and does not require any separation steps.
Jeong
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Caliper Technologies, Mountain View, CA, USA.
Nikiforov
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D019921:Biotinylation; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D019793:Fluorescein / Q000737:chemistry; D005453:Fluorescence; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*; D015203:Reproducibility of Results; D019809:Streptavidin / Q000737:chemistry
D009842:Oligopeptides; D010710:Phosphates; C022571:adenosine 5'-O-(3-thiotriphosphate); C037642:kemptide; D000255:Adenosine Triphosphate; D019809:Streptavidin; D004492:Edetic Acid; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D019793:Fluorescein; C035638:thiophosphoric acid