NI-NTA purification - (Apr/23/2010 )

Hello everyone,
Im using Sigma His Select Ni- NTA resin to purify one of my protein which i have solubilized using 8M urea. The purification protocol suggests use of either imidazole, or lowering the pH for elution of the his tagged proteins. Im not sure which one i have to follow.
The protocol says use of elution buffer at different pH to elute recombinant protein, i.e elution buffer with pH 4,5,6. Now in which order do i have to use the elution buffers,whether to start elution using buffer with pH 4,then 5, or 6, or in the reverse order, ph6, 5 and 4?.
Also will dialysis of the eluted fractions help in removing the urea from the recombinant protein?
pls help

you step away from the loading pH. so if you loaded at 7 then you would elute by trying 6 then 5 then 4...

you may want to try imidazole first.

yes, you dialyze away the urea (in steps or your protein will probably precipitate).

You have to bind your protein at pH 7,5-8 to a Ni resin due to the pI of the His tag, thus, you will elute it by decresing the pH step by step, the fact is that at so low pHs maybe your protein can be unstable, if you eliminate the urea at the same time you reduce so much the pH you can aggregate your protein, then i would use imidazol as eluent instead of reduced pHs.
If you perform your elution without urea, you will reduce your urea concentration in the protein solution but you probably will have some urea concentration. To assure that you have no urea at all in your final preparation you should wash intensely and slowly before (better with a linear gradient of urea, from 100% 8 M urea to 0% 8 M urea for example in 10-20 CV) in order to avoid aggregation