0 item(s)
View cart
You have no items in your shopping cart.
27
Jan
How to Strip, Clean, and Recharge Nickel Agarose Beads
A Practical Guide to Optimizing Resin Lifespan and Purification Efficiency
Nickel agarose beads are widely used for affinity purification of His-tagged proteins. With proper regeneration, these resins can be reused multiple times, significantly reducing purification costs while maintaining high performance.
Unlike standard agarose resins, nickel agarose beads require a specific regeneration workflow: stripping bound nickel ions, cleaning the resin, and recharging with fresh metal ions before reuse.
When should nickel agarose beads be regenerated?
After completing a purification, regeneration needs depend on your next step:
Simple Rinsing is enough if:
- Purifying the same protein.
- No EDTA or reducing agents were used.
- Rinse with dIH2O and store in 20% ethanol.
Full Regeneration is required if:
- Switching to a different protein.
- High concentrations of EDTA/reducing agents used.
- Binding capacity has visibly declined.
The Regeneration Workflow
Transfer the beads into a gravity-flow column. Solutions should be applied sequentially, allowing each to pass through completely before the next addition.
Figure 1. Sequential application of buffers for resin regeneration.
1. Nickel Stripping Buffer
Nickel ions must be removed to prevent reactions with harsh cleaning reagents. EDTA is used as a chelating agent to strip the metal.
Recommended Stripping Buffer Recipe:
- 20 mM Sodium Phosphate, pH 7.0
- 500 mM NaCl
- 50 mM EDTA
Figure 2. Molecular mechanism: EDTA stripping nickel from the agarose matrix.
2. Resin Cleaning Buffers
Once stripped, the resin is cleaned of contaminants using specific reagents. Crucial: Rinse with dIH2O between each reagent to avoid precipitation.
| Cleaning Solution | Target Contaminant | Contact Time |
|---|---|---|
| 1.5 M NaCl | Ionically bound proteins | 15–20 mins |
| 1 M NaOH | Denatured/precipitated proteins | 1–2 hours |
| 70% Ethanol / Isopropanol | Hydrophobically bound proteins | 15–20 mins / 1-2 hrs |
3. Nickel Recharging & Storage
Apply 1.5 CVs of 0.1 M Nickel Chloride (NiCl2), then rinse thoroughly with dIH2O. This step restores the resin's binding capacity.
Pro Tip: You can substitute nickel with cobalt ions here to achieve different selectivity for your next purification!
Storage:
Store in 20% ethanol at 4°C to prevent microbial growth. Before use, simply rinse out the ethanol with dIH2O.
Longevity & Performance
Most nickel agarose beads can be regenerated 3 to 5 times. Over time, the agarose matrix naturally degrades. If capacity remains low after a full recharge, it's time to replace the resin.
Biofargo team
