Hispur nickel nitrilotriacetic acid ni nta agarose

ICL activity was assessed as described previously [15 (link)]. Briefly, 3×10⁶E. coli cells or 6×10⁶Erwinia oleae cells pre-grown for 3.5 h in Dulbecco's modified Eagle medium (DMEM) with 25 mM HEPES (Invitrogen) were mixed with EDTA (1 mM) and 400 ng of linearized plasmid pUC19 DNA and the mixtures were incubated for 40 min at 37 °C. After pelleting the bacteria, the DNA was purified from the supernatant and analysed by electrophoresis on denaturing (40 mM NaOH – 1 mM EDTA) 1 % agarose gels. ICL activity of Erwinia oleae was also tested in the presence of 400 nM 6-histidine-ClbS, which was purified with HisPur nickel-nitrilotriacetic acid (Ni-NTA) agarose (Thermo Scientific) from a culture of BL21(DE3) strain hosting the plasmid pET28a-ClbS-His, as described previously [15 (link)].

BL21(DE3) cells hosting the plasmid pET28a-ClbS-His (27 (link)) were grown overnight at 37°C in 50 ml LB–0.4 mM IPTG (isopropyl-β-d-thiogalactopyranoside). Bacteria were lysed by sonication in a mixture containing 50 mM NaH₂PO₄, 300 mM NaCl, 40 mM imidazole (pH 8), 200 μg/ml gentamicin, 1 mg/ml lysozyme, and complete protease inhibitor cocktail (Sigma). The cleared lysate was incubated for 1 h with HisPur nickel-nitrilotriacetic acid (Ni-NTA) agarose (Thermo Scientific) in lysis buffer, washed with a mixture containing 50 mM NaH₂PO₄, 300 mM NaCl, and 60 mM imidazole (pH 8), and eluted in 250 mM imidazole buffer. The eluate was stored at +4°C before use at 400 nM in a 100-μl or 2.5-ml volume for culture experiments.