Pierce protease inhibitor edta free tablet

Human condensin complexes were purified as described previously (Kong et al., 2020 (link)). Briefly, the five subunits of human condensin I and II, sub-complexes, and Q-loop mutations and were assembled into biGBac vectors (Weissmann et al., 2016 (link)) to create baculovirus for protein expression in HighFive insect cells. Cell were lysed in condensin purification buffer (20 mM HEPES [pH 8], 300 mM KCl, 5 mM MgCl₂, 1 mM DTT, 10% glycerol) supplemented with Pierce protease inhibitor EDTA-free tablet (Thermo Scientific) and Benzonase (Sigma). Cleared lysate was loaded on to a StrepTrap HP (GE), washed with condensin purification buffer, and eluted with condensin purification buffer supplemented with 5 mM Desthiobiotin (Sigma). Protein-containing fractions were pooled, diluted twofold with Buffer A (20 mM HEPES [pH 8], 5 mM MgCl₂, 5% glycerol, 1 mM DTT), loaded on to HiTrap Heparin HP column (GE), washed with Buffer A with 250 mM NaCl, then eluted with buffer A with 500 mM NaCl. Finally, size-exclusion chromatography was performed using condensin purification buffer and a Superose 6 16/70 or increase 10/300 column (GE).