| Assay Method Information | |
| | Preincubation Relaxation Assay |
| Description: | In the preincubation assay, CPT concentrations were 50 and 100 μM, whereas various concentrations of compounds were 25, 50 and 100 μM. LdTOP1LS was preincubated with compounds 3a-d at different concentrations for 5 min at 37 °Cbefore the addition of substrate DNA. Relaxation assay was carried out as described previously with LdTOPILS serially diluted in the relaxation buffer (25mM Tris-HCl pH 7.5, 5% glycerol, 0.5mM DTT, 10mM MgCl2, 2.5mM EDTA and 150 mg/ml BSA) supercoiled pBluescript (SK ) DNA and 50mM KCl. The amount of supercoiled monomer DNA band fluorescence after ethidium bromide (EtBr) (0.5 mg/ml) staining was quantitated by using GEL Doc 2000 from Biorad (Hercules, CA, USA). |
| Affinity data for this assay | |
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