I don't think you need to be concerned. Do the purification without the reducing agent, or with the highest concentration that is tolerated by the resin, then add it afterwards to re-reduce the cysteines. The only drawback would be if you got disulfide crosslinking of different proteins to yours, causing them to copurify with the one you want, but this can be limited by working with a dilute starting material.

Yes. That's exactly what is happening. I am getting some other proteins along with protein of interest. Also, I tried dealing with it the way you suggested, that is, I started with low concentration of reducing agent and then increased the reducing agent concentration. However, that didn't seem to help.

Dear Shruti,

Most nickel resins will withstand up to 5 mM betamercaptoethanol without any problem at all and some can take up to 10 mM. Stay away from DTT as it a very strong reducing agent and it will transform your resin into a brown mess.

Ni-NTA resins will tolerate TCEP ((tris(2-carboxyethyl)phosphine) ) very well. Concentrations of 0.5-1mM should be adequate for maintaining free cysteines in a reduced state. TCEP is also more persistent than volatile reducing agents like BME and DTT, although it is not compatible with phosphate buffers. As others have mentioned, BME at up to 5mM is another option.

Dear Antonio & Oliver,

I have tried both BME as well as DTT. You rightly mentioned that Ni-NTA resin can tolerate 5 mM BME and when it comes to DTT, we cannot go beyond 1mM. However, I am still not able to purify the protein of interest. 

As suggested by Oliver, I am going to now try TCEP to see if I am able to get pure protein.

Thank you for your valuable suggestions. I will update you guys soon.

A 2nd purification step might be necessary. Ion exchange chromatography, hydrophobic interaction chromatography, and/or gel filtration chromatography can be used.

Hi,

I have tried other purification steps as well. But, since the yield for the protein of interest is not so good, I am trying if I can get pure protein after Ni-NTA only. Else, as you suggested, I may have to go for another round of purification step.

Hi,

Are you sure the contaminant is due to your proteins and not to non-specific binding to ni-nta? In case of contaminants binding the resin, have you tried using a cleavable His-tag?

Anyway, I have used BME up to 20mM with no prob. If the contaminant is mainly chaperones you can try washing twice the resin with 5mM ATP.

Thanks Paolo! I have been using TCEP as reducing agent. I have been able to purify the protein of interest using Ni-NTA finally but on small scale. I still need to standardise for large scale purification. 

A good resin for His purificaiton requiring EDTA or high amounts of reducing agents is the Roche cOmplete His-Tag Purification Resin. We have switched to using this resin almost exclusively - it never leeches any Nickel in EDTA or high amounts of DTT (even 10mM)

Thanks Andrew! We have recently ordered Ni-sepharose resin from GE and I am thinking of switching to that. Lets see how it works. Otherwise I can go for Roche one.