| Assay Method Information | |
| | Gel-Based Assay |
| Description: | Assay: Unless indicated otherwise, Tdp1 assays were performed in 20 ul mixtures containing 50 mM Tris-HCl, pH 8.0, 80 mM KCl, 2 mM EDTA, 1 mM dithiothreitol (DTT), and 40 ug/ml bovine serum albumin (BSA). For initial screening of Tdp1 inhibitors, 25 nM of 5'-32P-labeled substrate (D14Y) was reacted with 1 ng Tdp1 (~0.7 nM) in the absence or presence of inhibitor for 20 min at 25 C. Reactions were stopped by addition of 60 ul of gel loading buffer (98% (v/v) formamide, 1% (w/v) xylene cyanol, 1% (w/v) bromophenol blue). Twelve ul of aliquots were resolved in 20% denaturing polyacrylamide (AccuGel, National Diagnostics, Atlanta, Ga.) (19:1) gel containing 7 M urea. After drying, gels were exposed overnight to PhosphorImager screens (Molecular Dynamics, Sunnyvale, Calif.). Screens were scanned, and images were obtained with the Molecular Dynamics software (Sunnyvale, Calif.). Densitometry analyses were performed using ImageQuant 5.2 software package. |
| Affinity data for this assay | |
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