| Assay Method Information | |
| | LpxC Enzymatic Activity Assay |
| Description: | The experimental procedure is briefly described as follows: The test compound was first dissolved in DMSO to prepare a 10 mM stock solution. The reaction was carried out in a 96-well microplate. First, 20 μL of recombinant Pseudomonas aeruginosa LpxC (purchased from Signalway Antibody, product number AP74647-2) was added to each well, with a final concentration of 5 nM. 5 μL of the compound to be tested was then added, and the compound was diluted 4 times to 8 concentration points with a concentration range of 0.61-10,000 nM. 5 μL of LpxC substrate UDP-3-O—(R-3-hydroxydecanoyl)-GlcNAc (purchased from Biosynth Carbosynth, product number: mu75071) was added, with a final concentration of the substrate of 10 μM. The plate was incubated at 25° C. for 120 minutes. Then 20 μL of 2.0 mg/mL fluorescamine (purchased from sigmaaldrich, product number: F9015, solvent: 1:1 dimethylformamide/acetonitrile) was added to the reaction system, and mixed well for 10 minutes of reaction. Finally, 50 μL of 200 mM sodium phosphate buffer (pH 8.0) was added to terminate the reaction. Reading was carried out using a microplate reader (BMG), and the excitation and emission wavelengths were 390 and 495 nm respectively. |
| Affinity data for this assay | |
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