| Assay Method Information | |
| | In vitro Kinase Assay |
| Description: | In vitro kinase assays for c-Src variants were performed in 50 mM Tris (pH 8.0), 10 mM MgCl2 and 1 mg/mL BSA. When obtaining kinetic parameters (kcat, Km) kinase and peptide substrate (IYGEFKKK) (SEQ ID NO:51) concentrations were 2 nM and 500 μM, respectively, while ATP concentrations ranged from 2000-0.655 μM. Addition of nonradioactive ATP supplemented with 32P ATP (3,000 Ci/mmol, NEN) was used to initiate kinase reactions. Time points were selected such that product formation never exceeded 10%. Reactions were quenched by spotting 3 μL quantities onto phosphocellulose sheets (P81, Whatman). Afterwards, the sheets were washed 3×5 minutes in 0.5% phosphoric acid and dried. Radioactivity was measured by phosphorimaging and recorded on a Typhoon fluorescence imager (Molecular Dynamics). |
| Affinity data for this assay | |
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