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Assay Method Information |
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| | Gel-based Assay |
| Description: | A 5′-[32P]-labeled single-stranded DNA oligonucleotide containing a 3′-phosphotyrosine (N14Y) was generated as described by Dexheimer et al. The DNA substrate was then incubated with 5 pM recombinant Tdp1 in the absence or presence of inhibitor 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 the 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 Phosphorlmager screen (GE Healthcare). Gel images were scanned using a Typhoon 8600 (GE Healthcare) and densitometric analyses were performed using the ImageQuant software (GE Healthcare). |
| Affinity data for this assay | |
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