| Assay Method Information | |
| | Homogeneous Time-Resolved Fluorescence (HTRF) Assay |
| Description: | TrkAG667C: TrkAG667C (Kinase domain) kinase was expressed in Sf9 cells (purchased from Invitrogen) using pIEX-Bac-4 (purchased from Merck), and purified by using Ni column affinity chromatography on AKTA Purifier (GE company). A testing platform for TrkAG667C kinase activity was established based on Homogeneous Time-Resolved Fluorescence (HTRF) assay, and the activities of the compounds were tested using the platform. The compounds were subjected to five-fold gradient dilution with 100% DMSO with a starting concentration of 1 mM (8 concentrations in total). 4 μL of diluted sample for each concentration was added to 96 μL of reaction buffer (50 mM HEPES, pH7.4, 5 mM MgCl2, 1 mM NaVO3, 0.001% Tween-20, 0.01% BSA and 1 mM DTT) and mixed homogeneously to be used as a 4* compound. The reaction buffer was used to formulate 2* TrkAG667C kinases (the final concentration was 0.5 nM) and a 4* substrate mixture (ATP+TK peptide) (wherein, the final concentration of ATP was 15 μM; TK peptide, HTRF KinEASE -TK, was purchased from Cisbio, and the final concentration thereof was 100 nM) for use. 2.5 μL of the 4* compound was added to a 384-well plate (OptiPlate-384, purchased from PerkinElmer), and then 5 μL of the 2* TrkAG667C kinases were added, and mixed homogeneously by centrifugation. Then 2.5 μL of the 4* substrate mixture was added to initiate the reaction (the total reaction volume is 10 μL). The 384-well plate was placed in an incubator to incubate for 60 min at 23° C. Then the reaction was terminated by adding 5 μL of Eu3+ cryptate-labeled anti-phosphotyrosine antibody (HTRF KinEASE -TK, purchased from Cisbio), and 5 μL of Streptavidin-XL-665 (HTRF KinEASE -TK, purchased from Cisbio). After incubated for 1 h in the incubator, the fluorescence values were read on Envision (purchased from PerkinElmer). |
| Affinity data for this assay | |
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