| Assay Method Information | |
| | In Vitro Activity Test on TRKA, TRKB, TRKC Kinase |
| Description: | Table 1: Experimental MethodThe tested compounds were subjected to 3-fold serial dilution to reach a final concentration of 1 μM to 0.05 nM (10 concentrations), duplicates for each concentration; and the DMSO concentration in the detection reaction was 1%.TRKA Enzyme Reaction:0.2 ng/μL TRKA protein kinase, 1 μM TK Substrate-biotin polypeptide substrate, 14.68 μM ATP, 1×enzymatic buffer, 5 mM MgCl2, and 1 mM DTT. The assay plate was White Proxiplate384-Plus plate (PerkinElmer), and the reaction was incubated at room temperature for 40 min, and the assay volume was 10 μL.TRKB Enzyme Reaction:0.037 ng/μL TRKB protein kinase, 1 μM TK Substrate-biotin polypeptide substrate, 4.77 μM ATP, 1× enzymatic buffer, 5 mM MgCl2, 1 mM MnCl2 and 1 mM DTT. The assay plate was White Proxiplate 384-Plus plate (PerkinElmer), the reaction was incubated at room temperature for 50 min, and the assay volume was 10 μL.TRKC Enzyme Reaction:0.037 ng/μL TRKC protein kinase, 1 μM TK Substrate-biotin polypeptide substrate, 25.64 μM ATP, 1× enzymatic buffer, 5 mM MgCl2, and 1 mM DTT. The detection plate was White Proxiplate 384-Plus plate (PerkinElmer), the reaction was incubated at room temperature for 40 min, and the assay volume was 10 μL.Detection Steps:10 μL of detection reagent (containing 0.125 μM SA-XL665 and 5 μL 1×TK-Antibody) was added to the plate and incubated overnight at room temperature. Synergy Neo 2 was used to read the plate. |
| Affinity data for this assay | |
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