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| | TDP2 Assays with Whole Cell Extracts |
| Description: | Ten-million cells (1 x 107), either human, chicken DT40 wild type, or knockout for TDP2 and complemented with human TDP2, were collected, washed, and centrifuged. Cell pellets were then resuspended in 100 uL of CelLytic M cell lysis reagent (SIGMA-Aldrich C2978). After 15 min on ice, lysates were centrifuged at 12 000g for 10 min, and supernatants were transferred to a new tube. Protein concentrations were determined using a Nanodrop spectrophotometer (Invitrogen), and whole cell extracts were stored at -80 °C. The TY19 single-stranded DNA oligonucleotide containing a 5'-phosphotyrosine (see above) was incubated at 1 nM with 10-20 ug/mL of whole cell extract to obtain 30-40% cleavage in the absence or presence of 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 ug/mL BSA, and 0.01% Tween 20. Reactions were terminated and samples analyzed. |
| Affinity data for this assay | |
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