Pd minitrap g 25 desalting column

Rosetta2 cells were transformed with the corresponding pET24a-based plasmids containing the VH domain open reading frames and plated onto LB again containing 50 μg/mL kanamycin and 34 μg/mL chloramphenicol. Single colonies were grown on 4 mL TB media supplemented with 50 μg/mL kanamycin and 34 μg/mL chloramphenicol, at 37 ^oC. Once OD_600nm = 1.5–2, the temperature was lowered to 20 ^oC, 0.5 mM IPTG was added, and the culture was grown overnight, with agitation. The cultures were pelleted by centrifugation, and resuspended in 1.3 mL VH lysis buffer (100 mM HEPES pH 8.0, 500 mM NaCl, 10 mM Imidazole, 10% glycerol, 10U Benzonase (Merck), 10% n-Dodecyl ß-D-maltoside, 1 mg/mL lysozyme, 1 mM MgSO4, 1× protease inhibitor cocktail (Nacalai Tesque)), and lysed through three freeze-thawing cycles at −80 °C and RT. The lysate was centrifuged at 3320 × g for 20 min at 4 ^oC, and the supernatant was transferred onto a purification column containing 200 µL of Ni-NTA resin (Thermo Fisher), and incubated for 15 min at RT. The column was washed with 10 column volumes of VH wash buffer (20 mM HEPES pH 8.0, 500 mM NaCl, 25 mM Imidazole, 10% glycerol) and eluted with 5 column volumes of VH elution buffer (20 mM HEPES pH 8.0, 500 mM NaCl, 500 mM Imidazole 10% glycerol). The samples were immediately desalted into PBS using PD MiniTrap G-25 desalting columns (Cytiva).