Column regeneration should be performed when a different protein is being isolated or when there is a significant loss in the yield of protein. If the Nickel Chelating Resin loses its green/blue color the column needs recharging.
Recharge:
1. Wash the resin with 5 column volumes of a solution 20mM sodium phosphate supplemented with 0.5M NaCl, 50mM EDTA at pH 7.0.
2. Wash with 5 column volumes of distilled water to remove EDTA.
If you want to do full regeneration, continue these steps:
i. Elimination of ionic interactions: Wash in batch for approximately 20 minutes in a solution with 1.5M NaCl, follow with a wash with 10 column volumes of distilled water.
ii. Elimination of precipitated proteins. Wash in batch for at least 2 hours with a solution 1M NaOH, follow with a wash with 10 column volumes of distilled water.
iii. Elimination of strong hydrophobic interactions: Resuspend the resin in batch with 30% isopropanol and wash for approximately 20 minutes, follow with a wash with 10 column volumes of distilled water.
iv. Elimination of lipids: Wash in batch for 2 hours with a solution 0.5% of non-ionic detergent in 0.1 M acetic acid. Rinse away the detergent with approximately 10 column volumes of 70% ethanol, follow with a wash with 10 column volumes of distilled water.
3. Add 5 volumes of 0.1M nickel sulfate hexahydrate.
4. Wash with 5 column volumes of distilled water.
5. Add 5 column volumes of the binding buffer. The column is now ready for use.
NOTE: If storing the column for a while store at 4oC in 20% ethanol.
