First obvious thing to do is to completely remove the imidazole from the wash buffer.  Also perhaps the His tag is not accesible to the resin, this is obviously the worst case scenario.  If this is the case I believe that you have 2 choices:  make another construct where the His tag is located elsewhere and hope that it would be accessible to the NTA resin.  Or devise another purification route, look to see if their is a property of your protein that you can exploit.  For example ability to bind to a co-factor or unusual isoelectric point.  If nothing obvious comes to mind then ion exchange chromatography is usually the place to go.

Hope this helps,

Marc

Thanks for your suggestions Marc. complete removal of imidazole from wash buffer and elution buffer with 250mM imidazole will work out?

I agree with Marc. At 10 to 20 mM Imidazole your protein should bind and stay bound to the Nickel resin. It looks like the His-tag is burried, which also isn't a good news for the integrity of your protein as burial of the Tag can also mean unfolding/misfolding of your protein. The first thing I would try is to change the Tag position (as advised by Marc) from one extremity of your protein to the other.

Brindha,

You need to remove the imidazole from the binding and wash buffers, but you need to keep the imidazole for the elution buffer.  This may work out but no guarantee I am not too hopeful that this will work but it is indeed a last effort to use this construct via this purification strategy.  As pointed out by Yann-Vai a buried tag can mean improper folding.  You need to decide where you want to spend your time and/or money.

Hopes this helps,

Marc

Brindha,

You can try decreasing imidazole concentration in the wash buffer to somewhere around 5 mM and you can try some redox reagents like DTT into your protein solution so that even if his-tag is buried inside the protein, it might interact with nickel resin. You can also think of adding some extra aminoacids like glycine, one that less likely to effect folding of your protein, in between his tag and your protein's amino acid sequence. That can help his tag to be more reachable. 

Thanks for your suggestions. I think its better to change the his tag position from N terminal to C terminal and check the purification.

Brindha

You already have lots of good suggestions. But, one possibility is that the His tag is buried so in this case I recommend pETM30 vector (EMBL vector collection) which has a His-GST-Tev and makes life life much easier.

the second, as suggested by others could be a charge- or oxidation-related issue which you might need to look into (even the pH of imidazole stock that you use). As a general thing, it could be useful for the discussion to mention what sort of protein you're trying to purify like membrane protein, zinc finger protein, or a protein that tends to form higher-order structure under certain conditions...

The protein i am working on is the viral structural protein and I guess I have to concentrate on the pH of the buffers after the addition of imidazole.

If the tag is buried and you can afford to denature the protein (and renature it later in vitro), using urea or GuHCl in the buffer should solve the problem.

Another very important point was brought up by Patricia. Have you checked the pH of your buffers? They should be slightly basic (above 7.5, to be safe), otherwise the chromatography will fail.

Yet another possibility is that the tag is being cleaved off by some protease. Can you confirm that the tag is there by Western blotting the cell lysate with an anti-His6 antibody?

First of all, you may try the different buffer system with the varied pH condition and also ion strength of salt, they are the basic issue of binding capacity.

Secondly, if your resin can coding other metal ion, try Cooper or Cobalt, they are also optional for his-tagged protein purification.

Finally, ion exchange resins, sometimes they will be great choice and somehow have other plus advantage like removing endotoxin.

Good luck.

Vincent 

I also faced the same problem,, and my N- terminal His tagged protein used to co-elute at 40mM imidazole with other contaminants.

So, I solved the problem by incorporating an additional 6 His tag (i.e 18 nucleotides) at the C terminal end just after my protein sequence (before the stop codon) using SDM..  Now my protein elution starts at 150mM Imidazole!!

Try adding another 6 His tag on the other end of ur protein by insertion SDM of ur construct, hope it works for u as well!!

Thanks friends.First I will try with the buffer systems with different pH in denaturing conditions.