Regeneration of HisTrap Ni-NTA column

Abstract

Regeneration of GE HisTrap column by stripping and re-charging.

GE HisTrap column
Binding Buffer:
- 20 mM PBS pH 7.4
- 0.5 M NaCl
- 20 mM Imidazole
Stripping Buffer:
- 20 mM PBS pH 7.4
- 0.5 M NaCl
- 50 mM EDTA
distilled H2O
regeneration solution:
- 0.1 M NiSO4 or 0.1 M NiCl2
cleaning solution:
- 1M NaOH
optional extra-clean:
- 30% isopropanol for removal of hydrophobically bound protein
- (or: 0.5% detergent in 0.1 M Acetic Acid)
storage solution:
- 20% Ethanol

Prepare solutions
- all solutions can be stored in cold room or sterile-filtered at room temperature
Strip beads (remove Ni++ with EDTA)
- 5 CV H2o @ 20 ml/min
- 8 CV stripping buffer @ 10 ml/min (recommended 5-10 CV)
Clean-in-place (CIP)
- 4 CV 1M NaOH @ 15 ml/min (fill column)
- pause for contact time of 1h (recommended 1-2h)
- 10 CV binding buffer for pH adjustment Note: recommended cleaning for more hydrophobically bound protein is 0.1-0.5% non-ionic detergent in 0.1 M Acetic acid with a contact time of 1-2 h.
Recharge beads
- load 0.5 CV Ni-solution at reduced flow rate
- wash with 5 CV binding buffer
- wash with 5 CV H2O
Prepare storage
- put column into 20% EtOH