Akta pure 25 explorer

Full-length PAX3::FOXO1 (GenBank accession code: AAC50053.1) was cloned into pET104.1 DEST plasmid with a carboxy terminal 12 His tag and an amino terminal internal HA tag (Supplementary Fig. S1). Recombinant protein expression was induced by isopropyl-β-D-thiogalactopyranoside in Escherichia coli strain BL-21 (DE3). Cell lysates were prepared using the BugBuster MasterMix (MilliporeSigma catalog no. 71456-4). Purification was done using a HiTrap Chelating HP 1 mL column in AKTA Pure 25 Explorer (Cytiva). The column was washed with water, charged with 100 mmol/L nickel sulfate and washed again with water. The cell pellet was dissolved in the running buffer (20 mmol/L sodium phosphate buffer pH 7.4, containing 500 mmol/L NaCl, 40 mmol/L imidazole), which was also used for equilibrating the column before sample application and consequent wash. Recombinant protein was eluted from the column with a linear gradient to running buffer containing 1 mol/L imidazole.

Full-length PAX3::FOXO1 (GenBank accession code: AAC50053.1) was cloned into pET104.1 DEST plasmid with a carboxy terminal 12 His tag and an amino terminal internal HA tag (Supplementary Fig. S1). Recombinant protein expression was induced by isopropyl-β-D-thiogalactopyranoside in Escherichia coli strain BL-21 (DE3). Cell lysates were prepared using the BugBuster MasterMix (MilliporeSigma catalog no. 71456-4). Purification was done using a HiTrap Chelating HP 1 mL column in AKTA Pure 25 Explorer (Cytiva). The column was washed with water, charged with 100 mmol/L nickel sulfate and washed again with water. The cell pellet was dissolved in the running buffer (20 mmol/L sodium phosphate buffer pH 7.4, containing 500 mmol/L NaCl, 40 mmol/L imidazole), which was also used for equilibrating the column before sample application and consequent wash. Recombinant protein was eluted from the column with a linear gradient to running buffer containing 1 mol/L imidazole.