HIS-FUSION PROTEIN PURIFICATION |
|
Last Update: December 2006 |
|
|
|
| PREPARE SOLUTIONS | |
| 1. 1M Tris, pH 7.9 (1 L): | Mix 121.1 g of Tris base with 800 mL dH2O. Add ~42 mL cHCl. pH is temperature dependent: ~0.03 pH units per 1oC increase. Make sure it is at RT before making final pH adjustments. Add dH2O to 1 L (Autoclave) |
| 2. 2 M Imidazole | Mix 136.16 g of imidazole with 1 L dH2O |
| 3. 8X Binding buffer (200 mL): | Mix 4 mL of 2M imidazole (40mM), 80 mL of 10M NaCl or 46.75 g of NaCl (4M) and 32 mL of Tris-HCl, pH 7.9 (160mM). pH to 7.9. Add dH2O to 200 mL |
| 4. 8X Wash buffer (200 mL): | Mix 48 mL of 2M imidazole (480mM), 80 mL of 10M NaCl or 46.75 g of NaCl (4M), and 32 mL of Tris-HCl, pH 7.9 (160mM). pH to 7.9. Add dH2O to 200 mL |
| 5. 4X Elute buffer (100 mL): | Mix 27.23 g of Imidazole (4M), 20 mL of 10M NaCl or 11.69 g of NaCl (2M), and 8 mL of Tris-HCl, pH 7.9 (80mM). pH to 7.9. Add dH2O to 100 mL |
| 6. 4X Strip buffer (200 mL): | Mix 29.67 g of EDTA (400mM), 40 mL of 10M NaCl (2M), and 16 mL of Tris-HCl, pH 7.9 (80 mM). pH to 7.9. Add dH2O to 200 mL |
| 7. 8X Charge buffer (100 mL): | Mix 10.51 g of NiSO4 (400mM), with dH2O to 100 mL |
| PROCEDURE | |
| 1. Prepare protein extract as described (186. B-PER Bacterial Protein extraction reagent; 111. GST-protein fusion expression and purification) | |
| 2. Charge column (for every volume of settled Nickel resin): wash ethanol away with 3 volumes of dH2O, followed by 5 volumes of 1X Charge buffer and finally wash with 3 volumes of 1X Binding buffer | |
| 3. Add Protein extract in 1X Binding buffer (mix extract with appropiate volume of 8X Binding buffer) | |
4. Let resin mix with protein at 4oC in a rocker for >30 minutes |
|
| 5. Wash column with 10 volumes (1-2 times) with 0.5-1X Binding buffer | |
| 6. Wash column with 6 volumes (1-2 times) with 0.5-1 X Wash buffer | |
| 7. Elute bound protein with 6 volumes (or less) of 1-2X Elution buffer | |
| 8. Strip the Nickel and any remaining protein with 6 volumes of 1X Strip buffer | |