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Star Republic: Guide for Biologists

Purification of his-tagged protein --- HisTrap

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.