Ni-NTA stands for nickel bound to tris nitriloacetic acid, a metal chelator. Ni-NTA is IMAC technology, witch stands for Immobilized Metal Chelate Affinity Chromatography.

This whole branch of chromatography is basically immobilized EDTA (Ethylenediamine tetra acetic acid) derivatives. So, yes proteins are eluted off IMAC with EDTA, a metal chelator.

What is indeed strange is that your protein stuck to the column in the presence of 500 mM imidazole and could be eluted only with EDTA. Failure to elute in the presence of 500 mM imidazole could be due to precipitation of the protein on the column, but then one would need to assume that EDTA somehow solubilizes it. Another possibility could be that the protein stuck to the resin due to hydrophobic interactions, which could be promoted by the high ionic strength of the 500 mM imidazole elution buffer, and would then be desorbed by the stripping buffer, which presumably has a lower ionic strength.

For 6xHis-tagged proteins, elution with 500 mM imidazole is overkill; 250 mM is more than enough to desorb proteins bound by IMAC interactions. This suggests that your protein is retained on the column by some other mechanism than binding of the 6xHis tag to the nickel in the resin.

Dear Julian, please note that some closely related questions have been asked earlier on RG:

Can His-tagged proteins be eluted from Ni-NTA by low pH instead of imidazole?

https://www.researchgate.net/post/Can-His-tagged-proteins-be-eluted-from-Ni-NTA-by-low-pH-instead-of-imidazole

(5 answers)

Can I use EDTA and/or DTT in wash buffer and elution buffer used for Ni affinity chromatography?

https://www.researchgate.net/post/Can_I_use_EDTA_and_or_DTT_in_wash_buffer_and_elution_buffer_used_for_Ni_affinity_chromatography

(8 answers)

and

How can I elute the His-tagged protein in non-reducing condition without using an imidazole buffer?

https://www.researchgate.net/post/How-can-I-elute-the-His-tagged-protein-in-non-reducing-condition-without-using-an-imidazole-buffer

(54 answers)

It might be worth having a look at the answers given to those questions. In my experience it often pays off to search the "Questions" and "Publications" sections of RG to find closely related questions and access relevant literature references.

Good luck with your research and best wishes, Frank Edelmann

Pierre Béguin , will proteins with free sulfhydryls bind tighter to Ni than 6-his?

OT. Be prepared for a non-helpful answer to be bombed with upvotes from cult-like friends. (I expect at least 3).

To @Steigimur Stefansson: good question; it never occurred to me; I presume that it may reduce the Ni and make the column turn black, like when you put dithiothreitol through it; suppliers of IMAC resins advise against this, although Ni is less sensitive to recuctive agents than Co. But I don't know if if happens with SH groups in proteins.

Dear Pierre Béguin I'm not surprised that dithiothreitol reduces the nickel-NTA complex and makes the column turn black. Dithiothreitol is a stronger reducing agent than thiols and –SH functionalized proteins, because its oxidation leads to formation of a six-membered ring with an intramolecular disulfide bridge. This is why the oxidized from is energetically favored and very stable, while on the other hand dithiothreitol is a strong reducing agent.

Hi Pierre Béguin . Most of the lit. I've seen talk about protein -NH2 and =NH His groups binding to immobilized Ni++ coordinated with COO-. Proteins with more than one free -SH groups binding to immobilized Ni++ ???.

(OT. FTE currently has only 1 upvoting friend. This is not acceptable!, where are all his friends?)

Hello everyone and thank you for the helpful answers. Based on Pierre Béguin comments, would it be wise to try a 100mM EDTA elution instead of Imidazole in 0.5M (or less) NaCl? I am unsure as to what method/buffer I should try now for my next purification of this protein to make a reproducible method. @Steingrimur Stefansson my protein consists of 50% hydrophobic AA's and it has 7 cysteine residues towards the C terminal. I was thinking of also including 5mM 2BME in the buffers, but I am also worried that the protein will not be functional for downstream assays. Overall what would you advise to try next?

Protein purification is a highly empirical process. If you are satisfied by the yield and purity of the prep you got as described in your first post, and if it is reproducible, don't change anything. The only inconvenience is that you have to reload the resin each time with Ni.