| Assay Method Information | |
| | WecA Assay |
| Description: | UDP-Glucosamine-C6-FITC (2 mM stock solution, 0.56 L), MgCl2 (0.5 M, 4 μL), β-mercaptoethanol (50 mM, 5 μL), CHAPS (5%, 11.25 μL), Tris buffer (pH 8.0, 50 mM), undecaprenyl phosphate (4 mM, 1.4 μL), and inhibitor molecule (0-100 μg/mL in Tris buffer) were place 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 4 h at 37° C. and quenched with n-butanol (150 μL). Two phases were mixed via vortex and centrifuged at 10,000 xg for 3 min. The upper organic phase was assayed via reverse-phase HPLC. The organic phase (30 μL) was injected into HPLC (solvent: gradient elution of 85:15 to 95:5 MeOH/0.05 M aq. NH4HCO3; UV: 485 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 C55-P-P-glucosamine-C6-FITC was quantified to obtain the IC50 value. The IC50 values were calculated from plots of the percentage product inhibition versus the inhibitor concentration. |
| Affinity data for this assay | |
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