Fractogel emd deae column

Recombinant human HSPB1 was expressed in Escherichia coli BL21 (DE3) (Thermo Fisher Scientific, Waltham, MA, USA) cells by using the pAK3038Hsp27 plasmid [41 (link)]. The subsequent purification of the recombinant protein was carried out as described in Buchner et al. [42 (link)]. In brief, E. coli BL21 (DE3) cells harboring the pAK3038Hsp27 plasmid were grown in the presence of ampicillin (Sigma-Aldrich, St. Louis, MO, USA) to the desired optical density, then induced with 0.5 mM isopropylthiogalactoside (IPTG) (Sigma-Aldrich). The expression of HSPB1 upon IPTG induction was assessed by Western blotting (Figure S3A). Cells were incubated for 3 h upon IPTG induction, then harvested by centrifugation for 10 min at 2600× g and at 4 °C and lysed as described in [42 (link)]. After lysis, the proteins were precipitated with 35% ammonium sulfate and purified by ion exchange chromatography on a Fractogel EMD DEAE column (Merck Millipore, Burlington, MA, USA) using a 50 to 600 mM NaCl linear gradient. Eluted fractions were characterized for the presence of HSPB1 by SDS-PAGE and Western blot analysis (anti-HSPB1, SMC-161, StressMarq, Victoria, BC, Canada).