A-B Toxin

Purification of the chimeric fusion protein via Ni2+ affinity chromatography

Materials:

Binding buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea, 10 mM imidazole

Wash buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea, 50mM imidazole

Elution buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea, 250 mM imidazole

Cleansing buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea, 1 M imidazole

Protocol:

1. Resuspend the bead s and add 15ml ddH2O to per-wash

2. Add 15ml binding buffer to balance the column and flow out all the binding buffer

3. Close the flow valve , add 15ml supernatant to the column, incubate for 30min, stir the beads when the beads precipitate.

4. Wash with 5ml binding buffer

5. Wash with 10ml wash buffer

6. Close the flow valve, add 1ml elution buffer, stir by pipetting, incubate for 2min, collect the eluent

7. Repeat step 6 three times

8. Wash with 15ml cleasing buffer

9. Wash with 20ml 22% ethanol

10. Store in 3ml 22% ethanol