| Assay Method Information | |
| | MraY Assay |
| Description: | Park's nucleotide-N -C6-dansyl (2 mM stock solution, 1.88 μL), MgCl2 (0.5 M, 5 L), KCI (2 M, 5 μL), Triton X-100 (0.5%, 5.63 μL), Tris buffer (pH 8.0, 50 mM), neryl phosphate (0.1 M, 2.25 μL), and inhibitor molecue (0-100 μg/mL in Tris buffer) were placed in a 500 μL Eppendorf tube. To a stirred reaction mixture, P-60 (10 μL) was added (total volume of reaction mixture: 50 μL adjust with Tris buffer). The reaction mixture was incubated for 2 h at room temperature (26 oC) and quenched with CHCl3 (100 μL). Two phases were mixed via vortex and centrifuged at 25,000 xg for 10 min. The upper aqueous phase was assayed via reverse-phase HPLC. The water phase (10 μL) was injected into HPLC (solvent: CH3CN/0.05 M aq. NH4HCO3=25:75; UV: 350 nm; flow rate: 0.5 mL/min; column: Kinetex 5 μm C8, 100 Å, 150 ×4.60 mm), and the area of the peak for lipid I-neryl derivative was quantified to obtain the IC50 value. |
| Affinity data for this assay | |
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