| Assay Method Information | |
| | Inhibitory Activity of Compounds to ROS1-G2032R |
| Description: | Based on LanceUltra (Perkin Elmer, CR97-100) principle, ROS1-G2032R kinase activity detection platform was 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, were 3-fold serially diluted to obtain 11 samples for multiple hole inspection. TPX-0005 (WuXi AppTec. supplied) was used as positive reference compound. The gradient diluted compounds were mixed with 0.016 nM ROS1-G2032R kinase (Abcam, Cat. ab206012), 50 nM LANCE Ultra ULight-poly GT peptide (PerkinElmer, Cat. TRF0100-M) and 2.6 μM ATP (Sigma, Cat. A7699) in the Optiplate-384F plate (PerkinElmer, Cat. 6007299) and incubated at room temperature for 60 mins. 5 μl 40 mM EDTA was used to stop the reaction. Then 2 nM Europium-anti-phosphotyrosine (PT66) (PerkinElmer, Cat. AD0069) was added and incubated at room temperature for 60 mins. The LANCE signal was obtained by EnVision™ (PerkinElmer, 2014) (Excitation light, 320 nm; Emission light, 665 nm). |
| Affinity data for this assay | |
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