| Assay Method Information | |
| | Inhibitory Activity of Compounds on TRKA and ALK-L1196M |
| Description: | Based on HTRF of Cisbio (Cisbio, Cat. 08-52) principle, TRKA and ALK-L1196M kinase activity detection platform were established to determine the inhibitory activity of compounds. The compound powder was dissolved in 100% DMSO (Sigma, Cat. D8418-11) to prepare a 10 mM storage solution. The compounds had an initial test concentration of 1000 nM and 10,000 nM respectively, were 3-fold serially diluted to obtain 11 samples for multiple hole inspection. RXDX-101 (WuXi AppTec. supplied) or Crizotinib (WuXi AppTec. Supplied) was used as positive reference compound.The gradient diluted compounds were mixed with 0.5 nM TRKA (Carna, Cat. 08-186)/ALK-L1196M (Carna, Cat. 08-529), 0.3 μM/1 μM TK Substrate-biotin and 90 μM/30 μM ATP (Sigma, Cat. A7699) in a Optiplate-384F plate (PerkinElmer, Cat. 6007299) and incubated at room temperature for 90 mins/120 mins. Then 0.67 nM Eu-TK-Antibody and 50 nM Streptavidin-XL-665 were added, mixed and incubated at room temperature for 60 min. The fluorescence value was obtained by Envision (PerkinElmer, #2014) (Excitation light, 320 nm; Emission light, 665 nm). The IC50 values of the compounds were calculated using XLFIT5 (IDBS) software. |
| Affinity data for this assay | |
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