| Assay Method Information | |
| | In Vitro Evaluation of Inhibitory Activity Against Syk Protein Kinase |
| Description: | Test Procedure and Method:(a) Compound dilution and microplate injection1) Compound powder was weighed and dissolved in a specific amount of DMSO at an initial concentration of 10 mM.2) The compound was diluted to have a concentration of 0.74 mM, POD18 was used for microplate injection by 135 nL per well, an initial concentration of the compound was 10 μM, and 11 concentrations and 3-fold descending serial dilution were carried out.(b) Reaction stage of enzyme and substrate1) Preparation of test buffer dilution: 5×HTRF buffer in the kit was diluted to 1×HTRF buffer, and a specified amount of DTT and MgCl2 solution were added for later use.2) A Syk enzyme reaction solution was prepared with 1×HTRF buffer, so that a final reaction concentration of Syk kinase was 0.0156 ng/μL.3) A TK-Substrate-biotin/ATP mixture was prepared, so that a final substrate concentration was controlled to be 0.2 μM. An ATP concentration was controlled to be 2 μM.4) A mutidorp injector was used to add the sample, 5 μL of Syk enzyme solution and TK-Substrate-biotin/ATP mixture were added to each well, and incubated at 23° C. for 1 hour. |
| Affinity data for this assay | |
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