Sp sepharose fast flow matrix

N-terminal Flag-tagged hTDP1 alleles were expressed from YEpGal4-10GAL1-FLAGhTDP1·L vectors in protease-deficient top1Δ,tdp1Δ cells (ECY2 strain). Exponential cultures were induced with 2% galactose for 6 hours, cells were harvested, and FLAGhTDP1 protein purified as described in [32 (link)]. Briefly, cell extracts in HEE/2PI buffer (50 mM HEPES, pH 8.0, 5 mM EDTA, 5mM EGTA, 2PI [2xprotease inhibitor cocktail, Sigma]) were loaded onto SP Sepharose fast flow matrix (GE Life Sciences) and eluted with HEE/2PI/0.4 M NaCl, then affinity purified by anti-FLAG M2 affinity chromatography matrix (Sigma). FLAGhTDP1 was eluted with 3X FLAG peptide in TEEK (50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 100 mM KCl, 1 mM DTT) and concentrated in an Ultracel-30K concentrator (Millipore). Concentration was determined by Bradford-assay (Bio Rad) and purity was determined by sypro-ruby (Bio Rad) staining of hTDP1 fractions resolved by 12% SDS-PAGE.

N-terminal Flag-tagged yTdp1 and hTdp1 alleles were cloned into YEpGal4-10GAL1·L vector and transformed into ECY2. Transformed yeast cells were grown at 30 °C in selective media supplemented with 2% dextrose the first night, 1/100 diluted into selective media supplemented with 2% raffinose. Raffinose cultures were induced with 2% galactose for 6 h before harvesting. Cells were harvested and protein purified as described in Gajewski et al.^{21 (link)}. Briefly, cell extracts in HEE/2PI (50 mM HEPES, pH 8.0, 5 mM EDTA, 5 mM EGTA) were loaded onto SP Sepharose fast flow matrix (GE Life Sciences) and eluted with HEE/2PI 0.4 M NaCl, then affinity purified over anti-Flag M2 affinity matrix (Sigma). Flag-Tdp1 was eluted with 1 mg 3X Flag peptide in TEEK/2PI (50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 100 mM KCl, 1 mM DTT). Tdp1 collected in the flow-through and concentrated in an Ultracel-30 K concentrator (Millipore). Protein concentration was determined using Bradford-assay (Bio Rad). Purity was checked by 10% Bis–Tris SDS-PAGE followed by Sypro Ruby staining and immunoblotting.