Materials:

Equipments:

1. 1. Refrigerated centrifuge

2. Sonicator equipped with microtip

3. UV/visible spectrophotometer

Reagents and samples :

1. 1. Ni-NTA agarose resin slurry (commercially available)

2. The bacterial culture having over-expressed 6×Histidine-tag fusion protein

3. Hen egg white lysozyme

4. RNase A

5. Dnase I

6. 100 mM phosphate buffer, pH 7.4

7. 100 mM NiSO₄ ·6H₂O solution

8. 1 M imidazole solution

9. 1 M NaCl solution

10. 0.1 M EDTA, pH 8.0

11. 2% (w/v) SDS

12. Ethanol

13. 1 M acetic acid

14. 8 M guanidine·HCl

Glassware, plasticware, and other materials :

1. 1. A 10 ml capacity polypropylene column

2. Laboratory stand

3. Pipettes

4. Pipette tips

5. 200 ml volumetric flasks

6. 50 ml and 100 ml measuring cylinders

7. 1 ml and 5 ml glass pipettes

 

Preparation of reagents and matrix:

Binding buffer (20 mM phosphate buffer, pH 7.4 + 300 mM NaCl + 10 mM imidazole) :

1. 1. Take 40 ml of 100 mM phosphate buffer, add 60 ml of 1 M NaCl and 2 ml of 1 M imidazole solution.

2.  Add water to make the final volume to 200 ml .

Wash buffer (20 mM phosphate buffer, pH 7.4 + 300 mM NaCl + 25 mM imidazole) :

1. 1. Take 40 ml of 100 mM phosphate buffer, add 60 ml of 1 M NaCl and 5 ml of 1 M imidazole solution.

2.  Add water to make the final volume to 200 ml .

Elution buffer (20 mM phosphate buffer, pH 7.4 + 300 mM NaCl + 250 mM imidazole) :

1. 1. Take 40 ml of 100 mM phosphate buffer, add 60 ml of 1 M NaCl and 50 ml of 1 M imidazole solution.

2.  Add water to make the final volume to 200 ml.

Stripping solution (0.2 M acetic acid in 6 M guanidine·HCl) :

1. 1. Take 15 ml of 8 M guanidine·HCl solution and add 4 ml of 1 M acetic acid solution.

2.  Add 1 ml water to make the final volume to 20 ml.