| Assay Method Information | |
| | In vitro test of compound's inhibitory effect on Syk kinase (enzymatic experiment) |
| Description: | Table 1: Experimental steps and methods:a) Compound dilution and plate making1) The compound powder is weighed, dissolved in a certain amount of DMSO, and the initial concentration is 10 mM.2) Dilute the compound concentration to 0.74mM, use POD18 to plate, 135nL per well, the initial compound concentration is 10μM, 11 concentration points, 3 times decreasing gradient dilution.b) Enzyme and substrate reaction stage1) To prepare the experiment buffer dilution, dilute the 5×HTRF Buffer in the kit to 1×, and add the specified amount of DTT and MgCl 2 solution for later use.2) Prepare SYK enzyme reaction solution with 1×HTRF Buffer to make the final reaction concentration of SYK kinase 0.0156ng/μL.3) Prepare the TK-Substrate-biotin/ATP mixture so that the final substrate concentration is controlled at 0.2μM. The ATP concentration is controlled at 2μM.4) Use the Mutidorp sampler to add samples, add 5 μL of SYK enzyme solution and TK-Substrate-biotin/ATP mixture to each well, and incubate 23 solutions for 1 hour.c) Detection stage:1) Add 13.33mL of ethylenediaminetetraacetic acid solution to the detection buffer of the kit, add the specified amount of Eu-labeled antibody and XL-665, and prepare the detection solution.2) Use the Mutidorp pipette to add samples, 10μL of detection solution per well, and incubate with 23 solutions for 1 hour. Make it stop the reaction of the enzyme and substrate mixture.3) Read the value on Envision after centrifugation.d) Analyze the data: XL-Fit is used to analyze the data and calculate the IC 50 value of the compound. |
| Affinity data for this assay | |
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