Reduce the nickle with 2-mercapto EtOH or DTT to elute , but I agree with the first comment - your protein is not binding via His.

If your protein is in inclusion bodies it should be 100% misfolded, but also 100% pure. You can skip the column and just need to work out refolding from inclusion bodies.

If its not in inclusion bodies you might want to try showing the protein some B-Mercapto throughout lysis and binding in the presence of B-Me. Cobalt beads are a bit more resistant to reducing agents than nicklle, so maybe try Cobalt beads + 10mM B-Me to bind.

Last - change your construct. I've mutatated several Cys to Ser with spectacular results improving solubility and other biophysical behavior (intracellular protein).

Obviously the protein is not bound via His. You can try detergent for recovery, in this case urea/laurylsarcosine may work.

May be the concentration of imidazole used is not sufficient to elute your protein

You can try using high imidazol concentration (1M) with EDTA, and use long times for the elution, this may work. I had a similar problem, and this was my solution.

In myy PhD ahD thesis (please see on my profile), I experienced another problem: the protein didn't bind to NiNTA, despite clear Western detection with 6xHIS Ab. You might want to check out solubilizing agents of various modes of action (zwitterionic was working best for me), at different %, and at different protein:detergent ratios (see results in my thesis again).

Those hydrophobic proteins are truly pain in the neck!

Yes proteins tend to form inclusion bodies and also bind to strongly. I prepared a battery of different imidazole concentrations in EDTA and incubate at 37°C. After you find the most convenient Imidazole concentration you need to dialize the protein in re-naturing buffer to get some activity

Reduce the nickle with 2-mercapto EtOH or DTT to elute , but I agree with the first comment - your protein is not binding via His.

If your protein is in inclusion bodies it should be 100% misfolded, but also 100% pure. You can skip the column and just need to work out refolding from inclusion bodies.

If its not in inclusion bodies you might want to try showing the protein some B-Mercapto throughout lysis and binding in the presence of B-Me. Cobalt beads are a bit more resistant to reducing agents than nicklle, so maybe try Cobalt beads + 10mM B-Me to bind.

Last - change your construct. I've mutatated several Cys to Ser with spectacular results improving solubility and other biophysical behavior (intracellular protein).

Thank you all for answers. I am not a specialist in protein purification, and I have learned some important things through your comments. The immidazole concentration is not the problem as I have tried from 10 mM to 1M without any result. Yes, Laemmli buffer (SDS + B-Me) 5 minutes à 99°C enables protein to elute, and it could be a solution for me. Do you think it could be efficient without warming at 99°C??? The final goal is to send the protein to generate specific antibody against it.

When I extract my protein under native conditions it is in inclusion bodies, but under denaturing conditions it is solubilized. Whatever I can also try other detergent more zwitterionic.

Thank you again, I will inform you about the success of one or the other methods.

Richard

We get this with proteins that have aggregated on the column. You say it is applied in urea or another chaotrope. In these cases, we've always been able to elute with the chaotrope and EDTA (if using urea , make sure you have salt at 500 mM because the chelate resin is essentially a cation exchanger). Better yet, we use guanidinium HCl at around 3 - 6 M plus EDTA at say 10 mM , the column with turn white as the nickel and comes off along with your protein.

Which concentration of Urea has been used for dissolving of the protein sample?

And what was the condition of Chromatography? Native or denaturing? It is impossible to elute such a sample in native condition even with high concentration of Imidazole, EDTA etc.

Hello Richard

The proteins that are stored in inclusion bodies, habitually are sufficiently pure as for not need many purification steps and stiller if it will use to develop a polyclonal serum. I recommend to modify your purification protocol a little, since the one that you are using it seems not work properly. In my opinion it is not clear in what form your protein is in the resin and therefore whether it is or not bound to the same one.

I propose that you solubilizes the protein from the inclusion bodies with Guanidinio (6M) or Urea (8M) including mercaptoethanol and dialyze againt the same buffer you will use later for the nickel column. Then, you will be able to purify your soluble protein with the nickel column if it is not pure enough.

The conditions of elutions are 500mM NaCl, 8M Urea, in 20 mM Phosphate buffer at pH4. The addition of EDTA up to 100mM doesn't change anything.

Direct purification from inclusion body, OK, I can try it. Thanks a lot for answers.

I like what Jon Sayers said. I once used an affinity column for purifying polyclonal IgG against a target protein. Only very low affinity antibodies came off unless I used 6 M guandine HCl, pH 2. However, I eluted them very dilute into 0.1 M Tris HCl pH 8.4 so they would not permanently denature. In your case, I would add something to complex the nickel. At pH 2, EDTA and similar chelators are not soluble. Cysteine might be a good choice, concentration depending on the resin load.

I would suggest that you can try ion exchange or GPC after refolding the solubilised Inclusion bodies (IB) (refolding condition should be optimised). Since IBs are expected to be >80% pure, a single purification step after refolding might do your job.

If you are in a hurry to generate antibodies, the probable shortcut is to run the IBs on a low (6%) acrylamide gel and then excise the band from the gel, crush and mix well using syringe, emulsify with adjuvant prior to immunization. Some of my friends have used this technique to generate antibodies, that can be used for western. Since acrylamide is toxic, be careful to have the lowest possible acrylamide concentration

Still then one should prefer to generate specific antibodies immunising the functionally folded protein.

If your protein contains cysteine than you can add 2-5 mM beta mercaptoethanol in the solubilization buffer.

I had this problem and got round it by adding 0.5% triton-x-100 to the elution buffer. The protein that eluted was functional and very clean.

It has been stated that "proteins that are stored in inclusion bodies, habitually are sufficiently pure". This is partially true. Protein in inclusion bodies are generally already 95% or more pure. The problems come in the refolding.

You do not say how you re-fold your protein. This can be very tricky as you will probably get fractions containing: 1 - properly refolded protein, 2 - partially denatured protein, and possibly 3 - oligomers of the protein, 4 - mis-folded protein. The best way to sort this out is with a native gel. You will need some native properly folded protein to use for comparison. Once you confirm what the purity of your protein is with respect to the properly folded protein you can begin trying other denaturants to solublize your protein and different strategies for refolding.

As has already been suggested I would try some other expression system/construct to find one that does not produce the protein as inclusion bodies as refolding will be time consuming, possibly low yield, and can involve huge volumes in the dialysis if you are trying to make a significant amount of protein. We found this to be the case with aequorin... our dialysis step was against 25L of metal free buffer (aequorin binds calcium) and the dialysis had to be repeated at least 5 times to get a good yield of properly folded protein. After all that we still had to purify the protein by ion-exchange chromatography to separate the product from remaining mis-folded protein.

I want to know what is the maximum concentration of imidazole that could be used without affecting the nickel nta matrix is it good to use more than 2 molar concentration then what are the drawbacks of having such high imidazole concentration in protein please suggest

Strip the column with acid:

Protocol : https://cube-biotech.com/files/protocols/Washing_Regenerating_HisAffinityResins.pdf