Tomas, I have never been able to get IDA Cu-imidodiacetic acid or Fe-imidodiacetic acid to work under reducing conditions. I have also not heard of anyone else using it successfully under those conditions. For this reason, even though IDA is cheap and plentiful, the more expensive NTA is probably the best way to go.

Thomas, do you the reducing agent in there or can you use methionine cyeteine or more gentle one? we use IDA columns charged with Cu, Zn and other metals even for protein capture in some cases (1st step) from cell culture harvest. There are a bunch of chelators in cell culture and bacterial medias, and the cells secrete them as well, which if you add them directly to your pre-charged column will strip the metal; in the case of copper from blue to white. We have found that adding the appropriate amount of metal to the load will overcome this. We published a paper in B and B which if I knew how to send you from my Research Gate list, I could send you. if you can't acces this, please just drop me a note.

you cannot use Cu-IMAC under reducing conditions.

Thanks for the unpleasant answers :)

The thing is I just wanted to try this to see if my protein bound to the matrix and I got some reasonable purification. But I doubt my boss will allow me buy NTA just because it might work better.

The protein is already purified through Octyl Sep and HighQ columns, so there shouldn't be left any chelator. Anyway, I didn't even get to the point of loading my sample to the column, because it striped already when I washed it with the buffers. I guess I'll have to run it under non-denaturating conditions and add the bME immediately afterwards in that case.

Unfortunately my protein is little bitchy, one week it doesn't work under certain conditions, the other week it does and vice versa, so I spent half a year trying to keep activity for at least one week only to go back to my original conditions... The point is, I don't know whether methionine or cysteine would work, but right now, it seems that 1 mM DTT is highly inefficient while 15 mM bME works OK (unfortunately I didn't try other concentrations).

Did you consider using TCEP (tris(2-carboxyethyl)phosphine)? It is highly specific for disulfide bond and it does not reduces metals ions. 1 mM or a half of it should be sufficient.

all copper salts are blue in color, I once accidentally added protease inhibitor cocktail and the whole experiment was a failure. also we do not know what is your next step, if this protein is semi purified, then what is the purpose of cu-IMAC, since Cu_IMAC can bind proteins with even 1 histidine.

Tomas,

We manufacture and sell Cu-IDA resins and before buying other resin I would try the following.

I really don´t know if it will work with Copper but I know that works with Ni-IDA resins.

Please see below (I have copied the recommendation in Ni-IDA).

(*) Note: Under extended treatments with reducing agents, or in processes where high concentrations of these reagents are used, reduction of the metal ion may result – this will affect the binding capacity of the resin, so these agents should be avoided.

There is a very small loss of Nickel with this resin and it only happens in very drastic work circumstances. Given these conditions a slight brown discoloration may appear but does not usually affect performance. The discoloration is due to small particles of Nickel breaking away from the main body of resin and coming into contact with the reducing agents causing the Nickel to reduce and became brown in color. If there is a risk of producing this effect, it can be avoided by pre-treatment of the resin before it has ever been used. This treatment eliminates the cations that are weakly attached to the resin before beginning the process, so the reducing agent doesn’t affect the lost Nickel ions.

Pre-treatment:

1. - Wash the resin with five column volumes of distilled water.

2. - Wash the resin with five column volumes of binding Buffer (without reducing agents in the buffer).

3. - Wash with five column volumes of elution buffer (without reducing agents in the buffer).

4. - Equilibrate with 10 column volumes of binding buffer (without reducing agents).

Once this treatment has been carried out, the resin is ready for the simple purification in such circumstances as described.

Please, follow the recommended procedure with Cu-IDA resins and let me know if you get it. Probably, you will avoid to buy NTA but I cannot be sure about it in the case of copper.

Pilar

So, we didn't have to buy NTA since we already have it here with nickel :) I tried it under reducing conditions (5 mM bME) and it worked just fine (see below), then I recharged it with copper and it was striped, so I'll use the Ni-NTA.

@Sharad Purohit I'm trying to purify native enzyme with no His-tag on it, so I was just trying to see if there is some binding and I could use it for purification. Actually I got about 3-fold purification with 70% of activity recovered (another 7% was in unbound fraction).