| Assay Method Information | |
| | E. coli Topoisomerase I Relaxation Activity Inhibition Assay |
| Description: | The relaxation activity of E. coli topoisomerase I was assayed in a buffer containing 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.1 mg/mL gelatin, and 0.5 mM MgCl2. Half microliter from the appropriate stock solutions of compounds dissolved in the solvent (DMSO) or the solvent alone (control) was mixed with 9.5 μL of the reaction buffer containing 10 ng of enzyme before the addition of 10 μL of reaction buffer containing 200 ng of supercoiled pBAD/Thio plasmid DNA purified by cesium chloride gradient as substrate. Following incubation at 37° C. for 30 min, the reactions were terminated by the addition of 4 μL of a stop buffer (50% glycerol, 50 mM EDTA, and 0.5% (v/v) bromophenol blue), and analyzed by agarose gel electrophoresis. The gels were stained in ethidium bromide and photographed under UV light. |
| Affinity data for this assay | |
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