| Assay Method Information | |
| | Topoisomerase I Relaxation Assay |
| Description: | Supercoiled pHOT1 DNA (0.5 μg) was incubated with four units of human topoisomerase I in relaxation buffer (10mM Tris-HCl (pH 7.8), 1mM EDTA, 0.15M NaCl, 0.1% BSA, 0.1 spermidine, 5% glycerol), in the presence of varying concentrations of the test compounds. Reactions were carried out at 37 °Cfor 1 h and then terminated by the addition of sodium dodecyl sulfate (SDS) to 0.25% and proteinase K to 50 μg/ml. The reaction mixture was subjected to electrophoresis through a 0.8% agarose gel containing 0.5 mg/ml ethidium bromide in TAE buffer (40mM Tris-borate and 1mM EDTA). The gels were stained with ethidium bromide and photographed under UV light. For the quantitative determination of topoisomerase concentration activity, photographic negatives were scanned and the area representing supercoiled DNA, migrating as a single band at the bottom of the gel was measured using UVI-KS4000i gel documentation and analysis system (SyngenBiotech, Sacramento, CA). |
| Affinity data for this assay | |
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