| Assay Method Information | |
| | Enzymatic Assay for KpLpxH Inhibition |
| Description: | Briefly, two reaction mixtures were prepared. Mixture 1 contains 20 mM Tris-HCl (pH 8.0), 0.5 mg/mL BSA, 0.02% Triton X-100, 1 mM MnCl2, 1 mM DTT, 10% DMSO, and 200 UM substrate (UDP-DAGn), and mixture 2 is comprised of the same buffer, but instead of substrate, contains both LpxH (20 ng/ml) and 2× inhibitor. These mixtures were then pre-incubated at 37° C. for 10 min. To initiate the reaction, an equal volume of the LpxH mixture (mixture 2) was added to the substrate mixture (mixture 1) at 37° C. The final reaction solution contains 100 μM substrate, 10 ng/ml enzyme, and 1× inhibitor. At the desired reaction time points, an aliquot of 20 μL reaction mixture was removed and added to a well in 96-well half-area plate containing 5 mM EDTA (final concentration) to quench the LpxH reaction. Purified Aquifex aeolicus LpxE was then added to a final concentration of 5 μg/mL. The plate was incubated at 37° C. for 30 min followed by addition of formic acid to a final concentration of 3.75 M to quench the LpxE reaction. The malachite green reagent (Sigma Aldrich, catalog MAK307) was diluted 5-fold into the solutions, and the plate was incubated for 30 min at room temperature before the absorbance at 620 nm was measured. All measurements were done in triplicates, and standard error (S.E.) was calculated. |
| Affinity data for this assay | |
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