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| Description: | Agents: 2-fold kinase solution: using 1-fold kinase buffer to prepare 2-fold kinase solution, with a final concentration of 0.35 nM of BRAFV600E. 4-fold substrate solution: using 1-fold kinase buffer to prepare 4-fold substrate solution, with a final substrate solution concentration of 0.2 uM of Fluorescein-MAP2K1 and 1.5 uM of ATP. Compounds: The final test concentration of the compound was 10 uM at maximum. Firstly, a 100-fold concentration (i.e. 1000 uM) was prepared with 100% DMSO. The compound was diluted with 100% DMSO by 5 folds at 5 concentrations. The compound series was identical to that in the Mobility Shift Assay. After diluting with 1-fold kinase buffer by 25 folds, the resulting samples were shaked and mixed evenly on a plate-shaker for 10 mins. Procedure: To the 384-well reaction plate was added 4-fold compound dissolved in 10% DMSO at 2.5 l/well. To the 384-well reaction plate was added 2-fold kinase solution at 5 ul/well, and to the negative control wells were added 1-fold kinase buffer. The samples were shaked, mixed evenly, and kept standing at room temperature. To the 384-well reaction plate was added 4-fold substrate solution at 2.5 ul/well. The plate was reacted, shaked, mixed evenly and incubated at room temperature for one hour. Reaction result detection: 2-fold detection solution was prepared, with a final concentration of 2 nM of Antibody and 10 mM of EDTA. 10 ul of the detection solution was transferred to the 384-well plate to terminate the reaction. The plate was gently shaked on a plate-shaker for 30 mins. |
| Affinity data for this assay | |
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