| Assay Method Information | |
| | HTRF Kinase Activity Biochemical Assay |
| Description: | The reason was carried out in white low volume 384-well plate (Corning, USA) with 25 nL compounds in dimethyl sulfoxide in each well. To measure the compound mediated inhibition of kinase activity, 2.5 uL of the recombinant kinases were pre-incubated with test compounds for 30 minutes in the kinase reaction buffer (20 mM HEPES pH 7.4, 2 mM MnCl2, 10mM MgCl2, 100 uM Na3VO4, 0.0075% Triton X 100, 0.005% BSA and 1 mM DTT) prior to the addition of 2.5 uL of 1 uL if 1 uM biotin-leveled TK substrates and 10 uM ATP. Then the reaction was stopped after 1 hour incubation at room temperature by adding 5 uL of HTRF Detection buffer which also contains 0.375 nM Eu3+-cyptate-labeled TK antibody and 0.062 uM streptavidin-XL665(SA-XL665) to allow for detection of the phosphorylated peptide product. After 1 hour incubation at room temperature, the fluorescence intensity was measured with Envision plate reader (PerkinElmer, USA). |
| Affinity data for this assay | |
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