1. Wash the HisTrap column with 5 ml ddH₂O.

2. Load the column with 0.5 ml of 0.1 M nickel salt (NiSO₄).

3. Wash the column with 5 ml ddH₂O.

4. Equilibrate the column with 10 ml 20 mM sodium phosphate buffer (pH 7.5), 0.5 M NaCl, and 10 mM imidazole.

5. Apply the sample.

6. Wash the column with 10 ml 20 mM sodium phosphate buffer (pH 7.5), 0.5 M NaCl, and 10 mM imidazole.

7. Elute with 5 ml of 20 mM sodium phosphate buffer (pH 7.5), 0.5 M NaCl, and 500 mM imidazole. Collect 1 ml fractions.

8. Regenerate the column by washing with 10 ml of 20 mM sodium phosphate buffer (pH 7.5), 0.5 M NaCl, and 10 mM imidazole.