Dear all, I am using Ni-NTA agarose (Qiagen) for purification of His-tagged proteins by gravity-flow chromatography. Given that this agarose is remarkably expensive I would like to re-use it. For that purpose, could anybody kindly provide a good protocol for stripping-off (and reloading) Ni2+? After a quick research in the internet, I only found something for prepacked His-trap AKTA-columns but also read about some doubts that such protocols can be used for the Ni-NTA agarose for gravity-flow. I would appreciate any help. Thanks.
50 mM EDTA + 500 mM NaCl works great (NaCl helps to remove proteins aspecifically bound to resin).
Then wash extensively with H20 before reloading with Nickel
The NiNta manual by LifeTech specifies as follow :
To recharge 2 mL of resin in a purification column:
1. Wash the column two times with 8 mL 50 mM EDTA to strip away the
chelated nickel ions.
2. Wash the column two times with 8 mL 0.5 M NaOH.
3. Wash the column two times with 8 mL sterile, distilled water.
4. Recharge the column with two washes of 8 mL NiCl2 hexahydrate at a
concentration of 5 mg/mL prepared in sterile, distilled water.
5. Wash the column two times with 8 mL distilled water.
1. Add 0.02% azide or 20% ethanol as a preservative and cap or apply a
parafilm to the column. Store at room temperature.
Hello,
The nickel can be striped off using a chelator such as EDTA. I strip my Ni columns using a 20 mM TrisCl pH 7.9, 100 mM EDTA solution (in addition to any other components you have in your buffer). You can reload the column by using a few mL of a 50-100 mM NiCl2 solution. Qiagen have a resource with more details about this: www.qiagen.com/gb/resources/resourcedetail?id=79ca2f7d-42fe-4d62-8676-4cfa948c9435&lang=en. Hope that helps!
50 mM EDTA + 500 mM NaCl works great (NaCl helps to remove proteins aspecifically bound to resin).
Then wash extensively with H20 before reloading with Nickel
The NiNta manual by LifeTech specifies as follow :
To recharge 2 mL of resin in a purification column:
1. Wash the column two times with 8 mL 50 mM EDTA to strip away the
chelated nickel ions.
2. Wash the column two times with 8 mL 0.5 M NaOH.
3. Wash the column two times with 8 mL sterile, distilled water.
4. Recharge the column with two washes of 8 mL NiCl2 hexahydrate at a
concentration of 5 mg/mL prepared in sterile, distilled water.
5. Wash the column two times with 8 mL distilled water.
1. Add 0.02% azide or 20% ethanol as a preservative and cap or apply a
parafilm to the column. Store at room temperature.
The QIAexpressionist linked above is an invaluable reference for Ni-NTA. There are also much less expensive sources for Ni-NTA, with McLabs working very well in my experience and costing 1/6 the price of Qiagen. Both are Sepharose CL-6B based resin with NTA functional groups.
Dear Enrico König,
You can strip off Ni from the column by these simple steps: Pass 100mM EDTA (~20ml) and then pass 30ml MilliQ. Then again you can recharge the column with 100mM NiSo4 (~10) followed by 30ml MilliQ to remove extra unbound Ni from the matrix. Then equillibrate the column with binding buffer again or just store the column with 20% ethanol.
All the suggestions here sound quite convincing. What Nicolas suggested sounds to me like the best method, with only one addition; I would wash the beads at least 5-10 times with 8-10 mL water after washing with NaOH, since the basic pH (if still there) may cause detachement of the NTA-linker from the beads. I would also not store the column at room temperature but in the fridge.
Enrico, stripping and recharging conditions are pretty non-critical, in my experience. To avoid nasty surprises of nickel phosphate precipitation, make sure there is a water (or NaCl) gap between any use of phosphate based buffers and the application of nickel salt solutions. Once the column has been charged and washed, equilibration with PBS/N3 works fine.
Protocols are same. I like protocol for GE HiTrap columns.
But gravity flow colums have very low pressure and this is very bad if column content became too viscous during washing (e.g. in NaOH washing). In this case wash resin with mQ beyong column and repack it.
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