Regeneration of Ni-NTA beads

Abstract

Regeneration of Ni-NTA beads for protein purification

Ni-NTA beads:
- Ni-NTA Agarose from Qiagen (#30230) on Labhamster
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:
- 0.5 or 1.5 M NaOH
optional extra-clean:
- 30% isopropanol for removal of hydrophobically bound protein
storage solution:
- 20% Ethanol
SDS PAGE gel

Prepare solutions
- all solutions can be stored in cold room or sterile-filtered at room temperature
Strip beads (remove Ni++ with EDTA)
- spin down beads, discard supernatant
- stripping:
  - resusspend in 3 - 10 volumes stripping buffer
  - incubate 5 min rotating or shaking
  - optional: repeat once (spin down, discard, resuspend)
- wash two times:
  - spin down, discard supernatant
  - resuspend in 3 - 5 volumes of binding buffer
  - repeat
Clean beads
- spin down and resuspend in 3 - 5 volumes cleaning solution (0.5 M NaOH or stronger)
- incubate 10 min shaking or rotating
  - note: resin should turn white
- wash three times:
  - spin down and resuspend in 3 - 5 volumes distilled water
  - repeat twice
- optionally: wash & incubate with extra-clean solution followed by 3 x water wash
Recharge beads
- spin down and resuspend in 1 - 2 volumes of regeneration solution (Ni++)
- incubate shaking or rotating for 2 - 5 min
  - note: resin should turn blue-green
- wash:
  - wash twice with 3 - 5 volumes of distilled water
- equilibrate pH:
  - spin down and resuspend in binding buffer
Quality control
- boil small aliquot of beads in SDS sample buffer and run on gel
- stain well and look for left-over impurities
Prepare storage
- spin down and resuspend in 2 volumes of storage solution (20 - 30% Ethanol)
- label with original date and date of regeneration
- store in cold room