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Star Republic: Guide for Biologists

Purification of inclusion body proteins --- HisTrap

1. Harvest cells and resuspend in 4 ml 20 mM Tris-HCl pH 8.0.

2. Disrupt cells with sonication on ice.

3. Centrifuge and discard supernatant.

4. Wash the pellet with 3 ml cold 2 M urea, 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 2% Triton X-100 and sonicate.

5. Centrifuge and wash the pellet again with 2 M urea, 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 2% Triton X-100.

6. Resuspend the pellet in 5 ml 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 5 mM imidazole, 6 M guanidine hydrochloride, 0-5 mM beta-mercaptoethanol.

7. Stir for 30-60 minutes at room temperature.

8. Centrifuge at high speed at 4 C.

9. Transfer supernatant to a fresh tube and filter through 0.45 ml filter.

10. Wash the HisTrap column with 5 ml ddH2O.

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

12. Wash the column with 5 ml ddH2O.

13. Equilibrate the column with 10 ml 20 mM sodium phosphate buffer (pH 8.0), 0.5 M NaCl, 6 M guanidine hydrochloride, 0-5 mM beta-mercaptoethanol, and 5 mM imidazole.

14.. Apply the sample.

15.. Wash the column with 10 ml 20 mM sodium phosphate buffer (pH 8.0), 0.5 M NaCl, 6 M guanidine hydrochloride, 0-5 mM beta-mercaptoethanol, and 20 mM imidazole.

16. Change the buffer by washing with 10 ml 20 mM sodium phosphate buffer (pH 8.0), 0.5 M NaCl, 6 M urea, 0-5 mM beta-mercaptoethanol, and 20 mM imidazole.

17. Refolding by washing with a 30 ml 6-0 M urea gradient in 20 mM sodium phosphate buffer (pH 8.0), 0.5 M NaCl, 0-5 mM beta-mercaptoethanol, and 20 mM imidazole.

18. Continue wash with 5 ml 10 ml 20 mM sodium phosphate buffer (pH 8.0), 0.5 M NaCl, 0-5 mM beta-mercaptoethanol, and 20 mM imidazole.

7. Elute with 10-20 ml linear gradient 0-500 mM imidazole (20 mM sodium phosphate buffer (pH 8.0), 0.5 M NaCl, 0-5 mM beta-mercaptoethanol) or with 5 ml of 20 mM sodium phosphate buffer (pH 8.0), 0.5 M NaCl, 0-5 mM beta-mercaptoethanol, 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.