| Assay Method Information | |
| | DXR Enzyme Activity Assay |
| Description: | Oxidation of NADPH to NADP+ as a result of substrate turnover was monitored at 340 nm in a POLARstar Omega microplate reader (BMG Labtech). The standard reaction had a final concentration of 62.5 nM purified DXR protein, 0.5 mM NADPH, 100 mM NaCl, 25 mM Tris pH 7.5, 10% glycerol, 1 mM MgCl2 and 0.09 mg/mL BSA in 50 μL volume per assay. Reactions were initiated by the addition of DOXP after 15 min incubation of the reaction mixture without DOXP at 37° C. Absorption at 340 nm was measured continuously for up to 45 min. For Km [DOXP] determination, DOXP concentrations between 0 and 2 mM were tested at 0.5 mM NADPH. The linear range of enzyme activity was determined by varying the DXR concentration at 1 mM DOXP and 1 mM NADPH. IC50 assays were performed using the standard reaction conditions with the respective amount of DXR inhibitor added to obtain the given final concentrations. Data points from at least three independent replicates were analyzed by nonlinear regression using GraphPad Prism software. Slopes of changing absorbance values were converted to (μM DOXP)(mg enzyme)−1 s−1 using a NADPH standard curve (data not shown). For the determination of the inhibitory constant Ki [FSM] of DXR, enzyme activity over a range of DOXP substrate concentrations between 0 and 2 mM was measured for FSM between 0 mM to 4 mM. |
| Affinity data for this assay | |
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