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| | CYP51 Inhibition Assay |
| Description: | Briefly, the reaction mixture contained 1 uM CYP51, 2 uM cytochrome P450 reductase, 100 uM dilauroyl-alpha-phosphatidylcholine, 0.4 mg/ml isoctrate dehydrogenase, and 25mM sodium isocitrate in 20mM MOPS (pH 7.4), 50 mM KCl, 5 mM MgCl2, and 10% glycerol. After the addition of the 3H-radiolabeled sterol substrate (~2000 cpm/nmol, final concentration 50 uM) the mixture was preincubated for 5 min at 37 °C in a shaking water bath (GCA Precision Scientific). The reaction was initiated by the addition of 100 uM NADPH and stopped by extraction of the sterols with ethyl acetate. The reaction products were dried, dissolved in methanol, and analyzed by a reverse-phase HPLC system (Waters) equipped with a beta-RAM detector (INUS Systems, Inc.) using a Nova-Pak C18 column (particle size 4 uM, 3.9 x 150 mm) and a linear gradient consisting of water:acetonitrile:methanol (1.0:4.5:4.5) (solvent A) and methanol (solvent B) at a flow rate of 1 ml/min. Under these conditions the retention time for eburicol, obtusifoliol, and lanosterol was 26, 23, and 25 min, respectively. The retention time for the reaction intermediates, 14alpha-alcohol and 14alpha-aldehyde derivatives of obtusifoliol (30) (as seen in Fig. 3, L. infantum CYP51), was 10 and 12 min, respectively. |
| Affinity data for this assay | |
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