| Assay Method Information | |
| | AlphaLISA Assay |
| Description: | Table 8: Exemplary MEK inhibitor compounds (0, 0.01, 0.1, or 1 μM) were added to a 384-AlphaPlate (PerkinElmer, 6008350) by a digital liquid dispenser (Tecan D300e) based on a total volume of 20 μL reaction mixture. 5 μL 4×MEK1 stock solution was added to wells A1-P20 by multichannel pipettes. The plate was sealed with clear olefin sealing tape (Thermo Scientific, 232701), centrifuged at 1000 rpm for 1 minute, and incubated at room temperature for 30 minutes. 5 μL 4×CRAF stock solution at different concentrations or 5 μL or 10 μL AlphaLISA buffer was added by multichannel pipettes. The plate was sealed, centrifuged at 1000 rpm for 1 minute, and incubated at room temperature for 60 minutes. 5 μL 4×glutathione acceptor beads were first added to all wells by multichannel pipettes. The ambient light was subsequently adjusted to <100 lux (light meter) before adding 5 μL 4×nickel chelate donor beads to the same wells. From this step on, the plate was covered with a black plate cover, centrifuged at 1000 rpm for 1 minute, and incubated at room temperature for 60 minutes. |
| Affinity data for this assay | |
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