| Assay Method Information | |
| | Determination of the Compounds of the Present Invention on Activity of ALK Kinase |
| Description: | Table 4: The experimental flow was briefly described as follows: a test compound was first dissolved in DMSO to prepare a storage solution, and then diluted in gradient with a buffer provided in the kit. A final concentration of the test compound in the reaction system ranged from 1000 nM to 0.004 nM. An ATP Km value concentration of each ALK protein was determined by using an ATP solution diluted in gradient (Sangon Biotech (Shanghai) Co., Ltd., A600311). According to the Km value obtained, the ATP concentrations in the reaction system were set as 1 μM for ALK, 10 μM for ALK(G1202R) and 25 μM for ALK(L1196M, G1202R). The reaction was carried out in a 384-well microplate. Firstly, the compound and a certain amount of corresponding ALK protein were added to the wells and incubated for 5 minutes to 10 minutes at room temperature. Then, the ATP solution and the biotinylated polypeptide substrate solution were added to the reaction solution and incubated with shaking at room temperature for 60 minutes. Then, an anti-phosphorylated tyrosine antibody coupled with a europium compound and streptavidin coupled with modified allophycocyanin XL665 were added to the reaction, and continuously incubated with shaking at room temperature for 1 hour. After incubation, fluorescence intensity values of each well at an excitation wavelength of 304 nM, and emission wavelengths of 620 nM and 665 nM were determined in a TF-FRET mode on a microplate reader, and the fluorescence intensity ratio 665/620 of each well was calculated. |
| Affinity data for this assay | |
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