Assay Method Information
Assay Name:  Kinase Activity Assay
Description:  The principle of the activity assay was that TGFβR1 phosphorylates the substrate TGFβR1tide, consuming ATP in the process. This experiment employed the ADP-Glo method to detect kinase activity and determine the IC50 value, which was used to evaluate the inhibitory ability of the test compounds against human TGFβR1 In the experiment, DSM was used as a negative control, and LY364947 was used as a positive control. The specific experimental steps were as follows. Compounds were diluted 4-fold in DMSO in a dilution plate, with the final starting concentration of the compounds being 10 μM, across 10 concentration gradient points. The compounds were then further diluted 50-fold into the kinase reaction buffer and incubated on a shaker for 20 minutes. Kinase was formulated with an enzyme reaction buffer. 2 μl kinase was added to each well of the reaction plate. 1 μl of the compound diluted in buffer was added to each well, and then the plate was sealed with a sealing membrane and centrifuged at 1000 g for 30 seconds, leaving it at room temperature for 10 minutes. Solutions of TGFβR1tide and ATP were prepared with the kinase reaction buffer, and 2 μL of the TGFβR1tide/ATP solution was added to the reaction plate. The plate was sealed with a sealing film and centrifuged at 1000 g for 30 seconds. It was allowed to react at room temperature for 60 minutes. Subsequently, 4 μL of ADP-Glo was transferred to a 384-well reaction plate and centrifuged at 1000 rpm/min for 1 minute, followed by incubation at 25° C. for 40 minutes. Then, 8 μL of Detection solution was transferred to the 384-well reaction plate and centrifuged at 1000 rpm/min for 1 minute, after which it was incubated at 25° C. for 40 minutes. The RLU (Relative Luminescence Unit) signal was read using a BMG microplate reader, which was used to characterize the degree of kinase activity.
Affinity data for this assay
 
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