Measurement of the protein tyrosine kinase activity of c-src using time-resolved fluorometry of europium chelates
| Autor: | Donna R. Yarwood, Albert Braunwalder, Kenneth E. Lipson, Matthew A. Sills |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
chemistry.chemical_classification
Chromatography Kinase Biophysics Substrate (chemistry) Cell Biology Protein-Tyrosine Kinases Biochemistry Antibodies CSK Tyrosine-Protein Kinase Microtiter plate Substrate-level phosphorylation Enzyme src-Family Kinases chemistry Europium Isotope Labeling Phosphorylation Fluorometry Binding site Molecular Biology Proto-oncogene tyrosine-protein kinase Src Chelating Agents |
| Zdroj: | Analytical biochemistry. 238(2) |
| ISSN: | 0003-2697 |
| Popis: | A nonradioactive, sensitive assay method to evaluate the activity of protein tyrosine kinases is described. This method utilizes europium chelate-labeled anti-phosphotyrosine antibodies to detect phosphate transfer to a polymeric substrate coated onto microtiter plate wells. The amount of phosphorylation is then detected using time-resolved, dissociation-enhanced fluorescence. Recombinant c-src was used to demonstrate that substrate phosphorylation was dependent on incubation time, enzyme concentration, and the amount of substrate used to coat the microtiter plate wells. A series of proprietary c-src inhibitors was evaluated in competition assays, and demonstrated a rank order of potency which was identical to that determined by other assay methods. Substrate phosphorylation was also demonstrated to be dependent on the concentration of ATP present during the kinase reaction. Because the kinase assay can be performed with different ATP concentrations (unlike with assays utilizing radioactive ATP analogs), the assay described can be used to distinguish compounds that compete for the ATP or substrate binding sites of the kinase. |
| Databáze: | OpenAIRE |
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