Please suggest if I can reuse Ni-NTA column by recharging it with nickel sulphate, After elution of my protein with EDTA as it stripped Ni from the column.
Also suggest me what EDTA conc. normally used to elute His tagged protein like 200 to 500mM Imidazole use for elution?
please give the detailed protocol for that.
Thank you
Yes, you can recharge Ni-NTA columns using nickel salts (sulfate or chloride works fine). I strip the resin of Ni with 100 to 500 mM EDTA, then wash the beads with 1 N NaOH and 1% SDS, neutralize with a sodium phosphate buffer at pH ~7, and recharge with 100 to 1000 mM nickel chloride or sulfate. Make sure to wash thoroughly after the recharge, as any residual unbound nickel may compete for your histidine tag.
Unlike imidazole, where concentration is important, an EDTA elution can be thought as irreversible, meaning that with enough volume even very dilute EDTA solutions will completely remove Ni. The practical issues will be whether your protein remains soluble in EDTA, i.e. in the absence of divalent cations, and whether EDTA will interfere with downstream applications. Also, if your protein has limited solubility, a high EDTA elution may cause precipitation simply because you will elute in a smaller volume.
An elution buffer containing 50 to 500 mM EDTA is reasonable.
Yes, you can recharge Ni-NTA columns using nickel salts (sulfate or chloride works fine). I strip the resin of Ni with 100 to 500 mM EDTA, then wash the beads with 1 N NaOH and 1% SDS, neutralize with a sodium phosphate buffer at pH ~7, and recharge with 100 to 1000 mM nickel chloride or sulfate. Make sure to wash thoroughly after the recharge, as any residual unbound nickel may compete for your histidine tag.
Unlike imidazole, where concentration is important, an EDTA elution can be thought as irreversible, meaning that with enough volume even very dilute EDTA solutions will completely remove Ni. The practical issues will be whether your protein remains soluble in EDTA, i.e. in the absence of divalent cations, and whether EDTA will interfere with downstream applications. Also, if your protein has limited solubility, a high EDTA elution may cause precipitation simply because you will elute in a smaller volume.
An elution buffer containing 50 to 500 mM EDTA is reasonable.
I completely agree with Justin.
You can certainly recharge your Ni-NTA column.
Upon elution of protein with imidazole, I generally remove Ni with 50 mM EDTA. I then clean the column with 1 N NaoH followed by washing it with milli-Q water until the pH of eluent reaches as of milli-Q water. And finally I recharge it with 100 mM Ni-sulphate followed by washing with milli-Q water and equilibrating with the desired buffer.
Best,
Jogender
the question he is asking is if he can use the column after eluting with EDTA. All answers above are right when you elute and strip the column. here in this case, EDTA elution means you have Ni also in your elution buffer which is highly toxic divalent cation. If you have to get rid of it in further steps will be challenging and if you need to use it for therapeutic applications, you have reached a dead end. if you need EDTA to be in your elution, use EGTA instead which is similar to EDTA and specific for specific ions. It will not interfere also in elution. if you have to use EDTA, do not go more then 1 mM , even at this low concentration, there will be some leaching.
I wouldn`t prefer using EDTA in elution buffer when you use Ni-NTA col. However, there is a tolerable EDTA concentration recommended by GE health...
If there are some problems or concerns on EDTA, I would recommend using another system to remove metal cation from Ni-NTA – it is 100-200 mM citrate buffer with pH about 3.3-3.5 (in fact, citric acid solution with adjusted pH). Citric acid is a good chelator and removes Ni perfectly. And of course, then you can load Ni again and again.
We generally use an imidazole gradient of 50-500 mM to elute the recombinant protein from the Ni column and further washing the column with 500mM EDTA removes all the impurities from the column and makes it suitable for next use.
Answers from Justin W Chartron and Jogender Singh are correct. I follow the same protocol that they refer. I want to add that if the presence of EDTA in the final product could be a problem, you can use a simple gel filtration chromatography on a Sephadex G 25 column to remove it. In adition, it may be convenient if you are going to use any Cu-based method (Lowry or BCA) to estimate protein concentration. Chelating agents like EDTA produce negative interference witih this methods.
- Badges
- Science method