My recombinant protein of 38 kDa has been expressed and it was in soluble fraction (with 10% glycerol, 0.5mM IPTG and 23 degree celcius). but the protein was not binding to the Ni-NTA column. I suspect the his tag might have occluded within the protein tertiary structure. How can i purify my protein without adding any denaturants such as urea or Gdn-HCl.
Thanks in advance.
Thanks Dr. Adam,
Actually before going for the c terminal his tag, I want to optimize the condition where my protein can bind to the Ni-NTA matrix. So what kind of conditions can we do for optimising the binding of N-terminal His tagged protein to the column?
Hi there,
Before considering protocol optimization for NiNTA purification I would check the actual presence of the histag on the protein by WB.Then If present, tag accessibility might be improved by using non denaturing concentration of chaotropic agent or detergent.
Dear Dominique,
The western Blot shows negative but the SDS-PAGE shows the protein expression which is compared by uninduced culture as control. In solubility the protein is in both supernatent and pellet. Actually this was my truncated protein and the wild protein has no issues with the purification. But this truncated protein is not binding to the Ni-NTA. When I incubated the protein with 0.5M Urea the protein binds to the column and I need this protein in native state. If I refold the protein won't it form aggregation?
Hi again,
What I don't understand is that WB is negative but the protein is able to bind NiNTA in presence of urea... 0.5M is not a lot... So it might be that the protein is not denatured. In that case a simple dialysis after elution should be fine to eliminate urea.
The protein may be in the form of soluble aggregates, which occludes the His-tag. The low urea concentration may be sufficient to disrupt these aggregates. A non-ionic detergent may also be effective.
Did you have a positive control for the Western blot? Maybe the anti-His antibody is no good.
Hi Dominique,
Yes! I added 0.5M urea in the lysis buffer during the sonication and after the sonication centrifuged and purified as I did earlier and with this condition the protein gets eluted at 400mM imidazole elution buffer whereas without the urea treatment the protein gets eluted in was buffer (without imidazole), 10mM and 20mM imidazole elution buffers itself.
So can we say that this protein is in native condition? As you mentioned, Is simple dialysis is sufficient to refold the protein?
Dear Adam,
Yes I did WB with positive control with one of my his tagged purified protein. The purified protein has a positive band.
Hi again,
You can't conclude anything from the NiNTA experiment about the protein folding state. If the protein is pure enough at the elution stage you could run either a biological assay (if available) or a CD spec to check protein secondary structure content.
Dear Dr. Dominique,
Thanks once again,
I will check the biological assay of the protein
If the protein is oligomeric then can we check whether the protein is native or not through Size exclusion chromatography? If it shows the presence of oligomeric state can we conclude that the protein is native? I mean if it was molten globule can we detect through SEC as we know the mol.weight of the protein?
If protein is still under its oligomeric state it is under its native state. In case of denaturation, oligomer dissociation would have occured.
Thank you so much Dr. Dominique. I will dialysis and run SEC once. Thanks a lot for your expertise.
the reason your protein not binding to the column is may be degradation of its his-tag. check your sequence whether his-tag is in frame. or there is a stop codon before his-tag coding sequences.
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