| Assay Method Information | |
| | c-Cbl Biochemical Assay (TR-FRET) |
| Description: | Recombinant human c-Cbl (aa 47-435) was expressed in E. coli, purified and biotinylated in vitro. The protein was diluted to 12 nM in freshly prepared assay buffer consisting of 50 mM HEPES, pH 7.0, 100 mM NaCl, 5 mM MgCl2, 0.01% Triton-X 100, 0.01% BSA and 1 mM DTT. Recombinant human Src (aa 254-536)-GSSGSS-Zap-70 (aa 281-297) fusion protein was expressed in E. coli and purified. Protein was diluted in assay buffer to 5-20 nM and ATP was added to 1 mM. The HTRF assay signal was measured at 520 nm on an Envision plate reader, with reference signal at 485 or 620 nm. Data was normalized using maximum and minimum assay controls: % Inhibition=100−(100*((maximum control)−unknown)/(maximum control−minimum control)). A 4-parameter dose-response equation was used to fit the normalized dose-response data and derive an IC50 for test compounds. |
| Affinity data for this assay | |
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