Editing His-MBP Fusion Protein Purification Protocol (section)

== Ni column stripping and recharging  ==

<br>The Ni column does not have to be stripped and recharged between each purification if the same protein is going to be purified; it is sufficient to strip and recharge it after 5–7 purifications, depending on the cell extract, extract volume, target protein, etc. 

<br>Recommended stripping buffer: 20 mM sodium phosphate, 0.5 M NaCl, 50 mM EDTA, pH 7.4 

<br>Strip the column by washing with at least 5–10 column volumes of stripping buffer. Wash with 5 CV of 0.5 M NaCl and water, then at least 5–10 column volumes of binding buffer and 5–10 column volumes of distilled water before recharging the column. 

<br>Recharge the water-washed column by loading 2.5 ml of 0.1 M NiSO4 in distilled water on HisTrap HP 5 ml column. Salts of other metals, chlorides, or sulfates, may also be used (see “Optimization”).&nbsp; Wash with 5 column volumes distilled water, and 5 column volumes binding buffer (to adjust pH) before storage in 20% ethanol. 

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