| Assay Method Information | |
| | HTRF Kinase Activity Biochemical Assay |
| Description: | Table 3: For the SAR (structure-activity relationship) and compound screening, HTRF (Homogeneous Time Resolved Fluorescence) kinase activity assay was employed for all MER, AXL and TYRO3 kinases using Cisbio HTRF® KinEASE™-TK kit (Cisbio, USA). The kit includes biotin-labeled TK substrate, streptavidin-XL665, Eu3+-cryptate-labeled TK antibody and HTRF® Detection buffer. There are two main steps in the kinase assay: kinase reaction and detection of phosphorylated substrate. The reaction was carried out in white low volume 384-well plate (Corning, USA) with 25 nL compound in dimethyl sulfoxide in each well. To measure the compound mediated inhibition of kinase activity, 2.5 uL the recombinant kinases were pre-incubated with test compounds for 30 minutes in the kinase reaction buffer (20 mM HEPES pH7.4, 2 mM MnCl2, 10 mM 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 uM biotin-labeled 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+-cryptate-labeled TK antibody and 0.062 uM streptavidin-XL665(SA-XL665) to allow for detection of the phosphorylated peptide product. After 1 h incubation at room temperature, the fluorescence intensity was measured with Envision™ plate reader (PerkinElmer, USA). Upon excitation at 340 nm by UV, the energy from Eu3+ donor of the antibody is transferred to the FRET acceptor XL665, and XL655 emits light at 665 nm. The level of kinase activity was quantified by the HTRF ratio that calculated from the intensity of emission at 665 nm and emission at 620 nm (fluorescence intensity @ 665 nm/fluorescence intensity @ 620 nm×10,000). The recombinant protein of human MER (528-end) was purchased from Carnabio, Japan. The recombinant human AXL (473-end) and TYRO3 (455-end) were purchased from SignalChem, Canada. |
| Affinity data for this assay | |
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