| Assay Method Information | |
| | TDP1 Gel-based in vitro assay |
| Description: | Table 1 and 2: TDP1 Gel-based in vitro assay was carried out as previously described Lountos, G. T., et al. (Nucleic Acids Res. (2019) 47(19) 10134-10150.) (5′-Cy5-labeled DNA substrate (1 nM; N14Y; 5′-GATCTAAAAGACTT-pY-3′) was incubated with 10 μM recombinant TDP1 in the absence or presence of inhibitor (at concentrations ranging from 20 nM to 10 mM) for 15 min at room temperature in a buffer containing 50 mM Tris HCl, pH 7.5, 80 mM KCl, 2 mM EDTA, 1 mM DTT, 40 μg/ml BSA and 0.01% Tween-20. Reactions were terminated by addition of 1 volume of gel loading buffer [99.5% (v/v) formamide, 5 mM EDTA, 0.01% (w/v) xylene cyanol, and 0.01% (w/v) bromophenol blue]. Samples were subjected to a 16% denaturing PAGE and gels were exposed after drying to a PhosphorImager screen (GE Healthcare). Gel images were scanned using a Typhoon FLA 9500 scanner (GE Healthcare) and densitometric analyses were performed using the ImageQuant software (GE Healthcare). The IC50 of TDP1 inhibitors was calculated by comparing the percentage of cleavage product (5′Cy5-GATCTAAAAGACTT-p-3′) to DMSO control. |
| Affinity data for this assay | |
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