| Assay Method Information | |
| | Kinase Inhibition Assay (Inhibition of Enzymatic Axl Kinase Activity) |
| Description: | Compounds of Formula (I) as obtained above were initially diluted to 10 nM in 100% DMSO (CALBIOCHEM™) for storage and made into kinase buffer solution to create a compound concentration ranging from 1 uM and 10 uM. Serial dilutions of the compounds were dispensed into a 96-well plate (GREINER BIOSCIENCES™) at 6 μL each. Truncated human Axl (CARNA BIOSCIENCES™) were diluted in kinase buffer and added to the compound solutions and pre-incubated for 30 minutes at room temperature. Next, ATP (TEKNOVA™) and substrate solution (suggested manufacture substrates of PERKINELMER™, for example, ULIGHT™-TK peptide) for Axl (PERKINELMER™) was added (12 μL each) to the wells containing the compound solution and enzyme. The reaction mixture was incubated for 1 hour. Following the incubation, the stop solution made with EDTA, water, and Lance detection buffer (PERKINELMER™) were added (12 μL each) to stop phosphorylation. Following the addition of the stop solution and 5 minutes of shaking, the detection solution containing the Europium-labeled antibody (suggested manufacture substrates of PERKINELMER™, for example, PT66 for Axl), water, and Lance detection buffer were added (12 μL) to the reaction mixture and incubated again for 50 minutes. Substrate phosphorylation was a function of the 665 nm emission measured following the addition of the detection solution and 50 minutes of incubation. |
| Affinity data for this assay | |
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