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| | Recombinant TDP2 Assay |
| Description: | TDP2 reactions were carried out as described previously23 with the following modifications. A 18-mer single-stranded oligonucleotide DNA substrate (α32P-cordycepin-3′- labeled)23 containing a 5′-phosphotyrosine (TY19) was incubated at 1 nM with 25 pM recombinant human TDP2 (REC hTDP2) to obtain 30−40% cleavage in the absence or presence of an inhibitor for 15 min at RT in the reaction buffer containing 50 mM Tris-HCl, pH 7.5, 80 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 40 μg/mL BSA, and 0.01% Tween 20. Reactions were terminated by the addition of 1volume 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 with multiple loadings at 12 min intervals. |
| Affinity data for this assay | |
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