| Assay Method Information | |
| | Homogeneous Time-Resolved Fluorescence Assay |
| Description: | Homogeneous time-resolved fluorescence (HTRF) assays were performed in black, half-area, flat bottom, 96-well microplates (Costar #3694). Human mitochondrial LysRS with a C-terminal HA-tag (mLysRS-HA), diluted in HTRF buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM 2-mercaptoethanol, BSA at 1 mg/ml) was added (20 μl at a dimer concentration of 3.75 nM) in the wells of microplates placed on ice. Compounds of formula I or II (1 μl of a 10 mM solution in DMSO) were then added to the wells, and mixing was achieved by pipetting up and down 3-times 10 μl. After centrifugation at 900 g for 1 min at 4° C., plates were placed at 4° C. on thermal modules of an epMotion 5075 v automated pipetting system (Eppendorf). A 10 μl sample of IN-H6 diluted in HTRF buffer at a dimer concentration of 250 nM was added and mixing was achieved by pipetting up and down 3-times 15 μl. Incubation was conducted at 4° C. for 1 h. A 10 μl aliquot of a mix of antibodies prepared in HTRF buffer, directed to the His-tag, conjugated with Eu3+ cryptate (Cisbio #61HISKLB, 0.125 μl per test, prepared as recommended by the supplier), and to the HA-tag, conjugated with XL665 (Cisbio #610HAXLB, 0.375 μl per test, prepared as recommended by the supplier), was added and mixing was achieved by pipetting up and down 3-times 15 μl. Incubation was conducted at 4° C. for 30 min. |
| Affinity data for this assay | |
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