Other than imidizole, you can try eluting with EDTA as Ruud suggests (although co-eluting Ni-EDTA complex with your protein may cause other problems), or you can lower the pH of your buffer to elute your protein. How low? Well, the pKa of the imidazole side chain of histidine is ~6.0. So, at a pH significantly lower than that (say, pH 5), most of the histidines in the 6xHis-tag on your protein will be protonated, and protonated histidines will actually be repelled by the positively charged nickel ions in your resin. So, if your protein can tolerate the reduced pH, I suggest trying a block elution using a buffer at pH 4.5-5. Good luck!

Dear Zahir,

To prevent imidazole interference (usually with spectroscopic experiments) you can dialyze your protein into any buffer after the elution. This is a standard procedure. I think that even if you eluted your protein with something other than imidazole you could face the same problem - this something else interfering with your experiments.

Actually imidazole is contributing upto some extent with my results so thats why i can not use it further and want ot find a suitable solvent anybody used in experiment like EDTA but at the same time i want my protein in natural state.

Hi Zahir,

if your protein can take low pH, you can try elution with low pH (5,3 to 4,5). This will lead to protonation of the histidine residues, thereby eluting your protein of interest.

To protect your protein you could collect it in tubes containing a 10x concentrated buffer with neutral pH, to keep exposure to low pH as short as possible.

Another option would be to include a cleavage site between His-Tag and your protein and to do elution by digestion with specific protease like TEV for instance.

Best,

Kai

Try pH gredient.

You can try the decrease of pH as Kai Albring wrote, or elution by EDTA (that should not denature your protein, unless you have metaloenzyme?). But the simplest way is really to elute with imidazole and then remove by dialyzis or buffer exchange on Amicon.

I suppose that histidine would interfere with your measurements as well. You can try other chelating agents.

Yes Tomas, we are also expecting the interference of Histidines but we can have a control easily in that case. If it interfere then we can put a digestion site which could help us. I Think the low pH option would be better than the EDTA, because it would not require further steps.

Thanks to all of you for your valuable suggestion. It probably will help me.

If the samples contains a large number of contaminant molecules then pretreatment procedures such as dialysis or passing through amicon or PD10 would be useful to increase efficiency of binding of protein target to the column and elution with immidazole Using elution pHs lower than pK of histidine protonation (or gradient pHs) might be helpfull while making sure also that your protein  is stable at low pHs during purification process

Hi, you can also try Histidin amino acid in your elution buffer.

it will compete with your his-tag resin bond except that you will face some Ni leakage.

take care

yes, Firoozeh Salamat, you are quite right but we may have some problem with histidine as histidine also contain imidazole ring so we can not use it.

Can anyone clarify the non-imidazole buffer system at loh pH for histidine protein elution?

. Because not only the pH but also the composition of the buffer would be essential to elute the his-tagged protein from Ni-NTA-his column. I am waiting for your valuable suggestions

The choice should be done depending on your protein. Acetate buffer for example can be used between 4 and 6. But I wouldn´t go lower than 5.5. Again depends on your protein. Some can do well at low pHs and some not. Also consider a high ionic strenght to diminish inespecific interactions.

Barros-Alvarez, Could you specify the composition of acetate buffer along with ionic ingredients please?

Again, depends on your system but for ex. 25 mM sodium acetate, 500 mM NaCl, pH (you choose). But first bind your protein at a higher pH and wash to get rid of contaminants.

Yes, it may works. thanks

Hi Zahir,

check out this guide for expression and purification of His-proteins:

http://kirschner.med.harvard.edu/files/protocols/QIAGEN_QIAexpressionist_EN.pdf

You'll find a lot helpful protocols and recipes in there!

Best,

Kai

you could try elution with EDTA, followed by a gel filtration to remove Ni-EDTA if needed.

Other than imidizole, you can try eluting with EDTA as Ruud suggests (although co-eluting Ni-EDTA complex with your protein may cause other problems), or you can lower the pH of your buffer to elute your protein. How low? Well, the pKa of the imidazole side chain of histidine is ~6.0. So, at a pH significantly lower than that (say, pH 5), most of the histidines in the 6xHis-tag on your protein will be protonated, and protonated histidines will actually be repelled by the positively charged nickel ions in your resin. So, if your protein can tolerate the reduced pH, I suggest trying a block elution using a buffer at pH 4.5-5. Good luck!

Why don't you try to use glycine as a competitor. It is a weak competitor but should work. Good luck!

Dear, Boland, have you tried the Glycine? and what were the conditions e.g. pH, ionic strength etc

High concentrations of histidine will elute the protein, but it also be a problem with your downstream analysis. The solution though will likely depend upon what you want to do with the protein. Low pH will tend to strip the column so you will get increased amounts of metal ion in your sample. In many cases, this is worse then trying to get rid of imidazole (either by SEC or ion exchange chromatography). In that case you could try the new Roche nickle affinity resin, the metal ions are definitely more stable. In that regard, you can elute the bound His6-tagged proteins at much lower imidazole concentrations from this resin. In fact, we typically wash with 5mM Imidazole and elute at 50 or 100mM Imidazole.

If you lower the pH - you protonate the Histidine and you can remove your protein. Normally around pH 6 will do the trick

Tris/HCl may provide the positive charge needed to elute the His tagged protein.

A little addition to what everyone has already said: if you use mass spec on the eluted sample, remember to do at least a tip desalting.

If your construct is set up with a proteinase recognition site, just digest it off the column. I've done this with thrombin before, I swapped the column into a thrombin-friendly buffer, added thrombin, capped the column and then let it tumble gently overnight at 4 C. The next morning, I opened the stopcock, passed it over a disposable benzamidine column to remove thrombin, and I got very pure protein.

Dear, Fabrizio, could you elaborate the tip desalting for mass spec analysis?

Adding EDTA to the column should work (just make sure this EDTA solution has reasonable pH not to ruin your protein) - just be aware that it will most likely ruin the His-affinity column/resin: the lest you would need to recharge the resin if you want to reuse it, or will have to throw it away.

Dropping pH, as few people suggested already, may work if your protein tolerates this lower pH (around 5-5.5?). You can check pI of your protein, and if it is somewhere in this region, it may precipitate/aggregate or stick to the resin forever.

Another option is to reclone the protein with a different tag for purification, eg Strep-tag, or indeed introduce protease cleavage site, so you elute from the column by cleaving protein off.

Hi Zahir,

In addition to the suggestions above (EDTA, low pH, on-resin cleavage, different tag), you could remove the imidazole using a desalting column - just a type of size exclusion chromatography. Your protein will elute in the void volume while small molecules such as imidazole and salts, which have a higher retention time, will elute later. Depending on the initial volume there are a number of desalting columns available or that you can make. I use a gravity-flow PD10 desalting column (GE healthcare) to get rid of imidazole from NiNTA elutions. The other option is that you could dialyse it out. Depending on the physical properties of your protein it may precipitate when desalted or dialysed; there are no guarantees.

The method you will end up choosing is mostly dictated by your protein and a little bit of trial-and-error. Not all proteins withstand changes in pH especially if it crosses their isoelectric point (what is the pI of your protein). Not all proteins can withstand EDTA since some bind metal ions such as zinc fingers (does your protein have a structural metal ion?). And not all proteins can withstand certain protease cleavage conditions/may have pseudo-protease sites within them (does your protein have pseudothrombin cleavage sites?).

Good luck,

Soumya

Actually imidazole contribute to my protein result and we are not sure about the pure protein quality. thats why we can not use the imdiazole. We tried the dialysis process but perhaps it aggregate and denatured the protein which i want in native form. Now we are trying the GST-tag if we did not get the pure form from His tag

Can you please expand on what do you mean by "imidazole contribute to your protein result"? Do you have difficulty removing it? Is your protein metal-binding? Can you see protein precipitation/aggregating during the process of imidazole removal? More info may help to devise a strategy, and understand what the real problem is...

I have used a kit from Qiagen which uses Ni-NTA agarose and I detected my protein on western blot without any problems.

my protein aggregates during dialysis process in imidazole and also imidazole affect my results. I dont want imidazole for elution anymore. I am waiting for a valuable suggestion which any body tested himself or herself.

Zahir, have you tried using rapid desalting using a G25 column. You should be able to load up to ~25% of the column volume. This gets reduces the imidazole concentration rapidly, and, if it really is the imidazole that is your problem should help avoid the precipitation problem. Coupling this with the Roche's new Ni affinity resin which requires much less imidazole to elute should really help. Finally, why do you think that it really is the imidazole rather then Ni ions that are leaching from the column. In my experience (this will certainly be protein dependent and thus it does not mean its your problem) the metal ions are more likely the culprit. In this regard, the Roche resin is a better bet, since much less leaching. One drawback, is that for some proteins we have found the resin to have a much lower capacity so we continue to use the more traditional Ni resins for these. You should also know that as the resin ages you find more leaching of Ni. So if you need to use your current resin for whatever reason and it is the Ni rather then Imidazole you may not want to recycle the column to often, but instead re-pour the column more often. The desalting column will also help remove the metal ions, however, since they bind to the His tag they are harder to get rid of.

Cheers

John

Histidine instead of imidazole

Histidine, EDTA, or even metals, very high salts, various pH

Urea can be used to purify in denaturing conditions.

To desalt, you can use dialysis, ultrafiltration.

If the protein precipitate, you can extract in urea and slowly decrease urea concentration for renaturation.

You should also check the PI of the recombinant protein, and make sure you extract at pH at least one unit apart from PI

If your protein require imidazole or bind metals, alternative tags should be considered. They are plenty, GST is one, but if you need small tag, Strp tagII desbiotin elution is a good one to try, alternative using tags such as Flag, and purification with anti-Flag columns. Some companies such as Invitrogen, or qiagen, or GE, will have all those possible columns.

You can use descending pH gradient to elute your protein, I saw anyone suggest EDTA instead of imidazol but you should consider that EDTA would stripe the resin and you can't reuse it.

If your protein is soluble and you don't need a large quantity of it, you can try purification using an anti-His beads and His elution peptides. You can get these materials from MBL: http://ruo.mbl.co.jp/e/dtl/P/3311/. I used the method in the past and worked well in my hands.

Sarah, you are right that you will strip the resin. However, it is relatively trivial to recharge the resin with 0.5M NiCl2. We do this all of the time and get a pretty long run out of our columns. Eventually, the resin does break down. This statement is valid for the GE and most other resins. There may be one or two (Roche) where this is not true but in general, you are always losing NiCl2 (leaching) and it is good to recharge it occasionally.

Everything was already said here, great !

Just a comment about the use of histidine containing buffer. Surprinsingly, in our hands, histidine was less efficient than imidazole for elution. We had to use slighltly higher concentration to manage to recover the protein (i.e. our protein which was eluted at 200-250 mM Imidazole, was eluted at about 350-400 mM His !). Does somebody observe the same things ?

Beware with the possible inhibitory effect of high Ni concentration if you use nickel for elution.

I think the best way is to elute your sample by using another pH for your buffer to change the ionization of the histidine residues.

Looking again on your question, I just realized that your problem is probably not to find another way to elute your protein but that your real problem is to get rid off NI2+ ion and imidazole after elution. John Flanagan was right when he suggested to you to try to eliminate ions and imidazole by using a G25 size exclusion column. PD-10 column from GE Healthcare are ready to use. For a better result, you can equilibrate this G25 column with an EDTA containing buffer to allow efficient withdrawal of Histidin bound nickel ions.

What about Histidine instead of Imidazole?

Hi Gregory that was suggested earlier in the thread. Histidine does not work as well as imidazole, and depending upon how many His tags are available (for example on a monomeric vs oligomeric proteins) you may not get it off. Further, imidazole and histidine have similar chemical properties, and, thus may likely have the same effect. As was mentioned by Karl, you can lower the pH. However, not only will you affect the histidine pK's but the columns as well and your Ni ions will tend to be stripped from the column. As nickle can affect the oxidation states of Methionine and Cysteine you may not want this in your sample. There is a great deal of literature to suggest that these can cause downstream problems with some, but not all proteins. In principal, you could try including some Zn ions in your lysis buffer. This will certainly displace the His-tag, however, some proteins readily precipitate with Zn. Co is another choice but same issues as with Ni (also both Zn and Co could displace your Ni). It is possible that the Roche Nickle affinity column is more pH stable. Certainly the leaching of Ni ions is much much lower than the GE or others that I have tried. The Ni is better chelated by the resin consequently its less accessible for the His tags and requires less imidizole for displacement. It is possible that, if you are interested in pH, to displacement it would be a good choice.

You can use Histidine instead of imidazole.

in which vector do you did cloning the protein ? -----the his-tag....be in N-terminal or C-terminal ? what pI of protein ?