| Assay Method Information | |
| | Aminoacylation Assay |
| Description: | Compounds were solubilized in DMSO. Serial 2-fold dilutions covering two concentration ranges, 10 mM to 19.5 μM and 100 μM to 195 nM, were prepared. 0.6 μl/well of the diluted compound solutions (50× the final assay concentration) were added to white 384-well polystyrene assay plates (Thermo Fisher Scientific/Matrix Technology Corp., Hudson, NH). Uninhibited control wells (MAX)received 2 μl of 30% (v/v) DMSO. Baseline wells (MIN) received 2 μl of a solution containing 30% (v/v)DMSO and 15 mM L-phenylalanine (Sigma-Aldrich). Assays were performed in a buffer consisting of 50 mM Tris-HCl (pH 8.0), 50 mM NH4Cl, 10 mM MgCl2, 2 mM DTT, 0.005% Tween 20 (Surfact-Amps-20, Thermo Fisher Scientific/Pierce Protein Research products, Suwanee, GA), and 0.1 mM EDTA-NaOH (pH 8.0). Compounds were preincubated with 15 μl/well of either 2 nM E. coli PheRS, 2 nM H. influenzae PheRS, or 0.8 nM P. aeruginosa PheRS in buffer for 30 min at 2× final assay concentrations. The reactions were initiated with 15 μl/well of a 2× substrate solution in buffer containing 2 μM E. coli phenylalanine tRNA (Sigma-Aldrich), 100 μM ATP, and 2 μM [3H]Phe with a specific radioactivity of 6.3 Ci/mmol (PerkinElmer Life Sciences). The reactions were quenched after 30 min with 15 μl/well of a solution containing 4 mg/ml PVT/PEI/WGA type A SPA beads (PerkinElmer Life Sciences), 262 mM sodium citrate (pH 2.0), and 150 mM NaCl. The plates were sealed with transparent film (PerkinElmer Life Sciences). The beads were allowed to settle for a minimum of 2 h before scintillation counting with a Top-Count plate reader (PerkinElmer Life Sciences). [3H] counts (CPM) for aminoacylated tRNA were measured for 1 min/well. |
| Affinity data for this assay | |
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