The his tag is of 12 His residues at the C-terminus of the protein without a linker. So, what should be considered besides points of the typical protocol? Higher imidazole concentration of the loading and elution buffers? Higher/lower salt concentration? Co vs. Ni NTA resin?
With 12 His residues you "should" get higher specificity if all the His residues are available for binding to the metal. So yes, you should be able to use higher [imidazole] during binding, and cobalt would be a good choice (more selective). The other question is do you want to purify with a reducing agent and/or EDTA, since there are resins that have high tolerance for them and having 12 His will only help since they have weaker binding (Clonetech Talon for low levels of reducing agent but no EDTA, and Roche complete resin for reducing agents and EDTA). The key thing I found (see my posts on this) is that you want to saturate binding of your protein to the resin such that you have a protein/resin ratio where ~25% of your protein "does not bind", this will help to reduce non-specific proteins binding to the resin.
I would also do some pilot studies in batch mode with ~20 uL of resin in 1.5 mL of buffer using microfuge tubes to sort things out. I know Roche was offering free samples last year.
Thanks for a comment. At first glance, it seems that the protein-His12 bound to Ni-resin so tightly that I could not wash it with moderate concentrations of imidazole (up to 500 mM !) and higher concentrations of imidazole denatured and/or aggregated the protein. Thus, even 100 mM EDTA could not get the protein out of the column. Any ideas to elute 'protein-His12' at milder conditions to keep it intact?
One problem I have had with some proteins is that they stick to the resin through hydrophobic interactions and most does not come off. You could lower the pH to ionize the protein (calculate the protein isoelectric point: http://web.expasy.org/compute_pi/), add glycerol (10-20%) and detergent (NP40 0.5%) to the lysate and buffers. If you boil 15 uL the resin in 40 uL SDS/PAGE sample buffer and load it all onto the gel can you see the protein in the gel?
Like I said above, I would start the experiments in batch mode, saves a lot of time/reagent.
Nickle is a strong binder, going to cobalt will weaken the interaction.The Roche complete His tag resin (Co) is the weakest binder (so the most specific for a His tag) on the market that I am aware of, you could try that. If the main interaction is hydrophobic then high salt will only increase the binding of the protein to the resin versus the metal.
Hi,
Thanks for contributions, Gary and Alim. I think convergence of opinions of two experienced researchers says for itself and warrants my further trials.) I especially like a suggestion on boiling aliquot of resin from the top of the column and common advice to check different conditions in a small scale. Acidification and adding of glycerol/tween also will be considered.
To Alim. I also surprised when I got this stuff from a colleague, "why there are 12 of them, indeed?" =) I guess, this was because the source of the protein is cyanobacteria and there are lots of endogenous His-rich stuff inside them, but I can mistake.
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