Water → Imidazole-4-acetic acid

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반응 단계

A 10/2.5 cm Econocolumn (Bic-Rad, Hemel Hempsted, UK) was packed with 40 ml chelating Sepharose fast flow (Pharmacia Biotech, St Albans, UK) and equilibrated under gravity with 100 ml water. Metal ions (100 ml) were loaded as 0.1M copper sulphate, zinc chloride or nickel chloride (Sigma) in water and washed through with the same volume of equilibrium buffer (PBS/1M NaCl). NaCl was added to the concentrated dialysed supernatant to a final concentration of 1M to prevent leaching of metal ions from the column33. Up to 300 ml supernatant was loaded and the unbound material collected. Competitive elution was carried out using an imidazole gradient of 40, 60, 80, 100 and 120 mM, collecting 250 ml batchwise of bound product at each step. The column was regenerated by stripping metal ions with 100 ml of 50 mM EDTA and re-equilibrated with several column volumes of water.

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