| Assay Method Information | |
| | Enzymatic Assay |
| Description: | The inhibitory potency of compounds of Escherichia coli UDP-2,3-diacylglucosamine hydrolase (LpxH) was determined in an enzymatic assay. UDP-2,3-diacylglucosamine (UDP-DAG) was purified from the Caulobacter crescentus LpxI D225A mutant protein that contained a tightly bound UDP-DAG molecule. The enzyme was diluted using assay buffer containing 50 mM NaCl, 20 mM Tris-HCl pH7.5, 2.5 mM MnCl2, 0.01% Triton X-100.1 mg/mL BSA, and the final concentration is 2 nM. The compounds were diluted by Agilent liquid handler Bravo, the compound's serial dilution dose ranges from 100 μM to 1.7 nM. Then the enzyme and compounds mixture were incubated at room temperature for 10 mins. The enzymatic assay was started by adding UDP-DAG (FAC is 5 μM) and incubated for 20 mins at room temperature. The plate was then heated to 95° C. for 15 mins on a water batch to stop the reaction. The hydrolysis of UDP-DAG by LpxH yielded 2,3-diacylglucosamine 1-phosphate (lipid X) and UMP, which was converted to ATP and quantified by the luciferase reaction using the UMP/CMP-Glo™ Glycosyltransferase Assay kit from Promega. The compound's inhibitory effect of LpxH is determined by measuring the light change using a luminometer (Envision). |
| Affinity data for this assay | |
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