I have to purify a protein with 6xHis tag, but at the time of dissociating the protein from the resin (Ni-NTA) all protein remains in the resin. A feature of my protein is that it is rich in histidine in some sectors. I increased the amount of up to 500 mM of imidazole but I have not had good results. In advance thanks for the help.
As per the info you have given, you protein of interest is having high affinity for Ni-NTA beads owing to His-tag and some internal histidine rich sites. In this case i would suggest using Ni-NTA beads with lower binding capacity (Roche Ni-NTA beads), i.e with fewer sites of binding, which will help you while elution of bound proteins. and plus point is high affinity of your protein, which will make sure that only your protein will bind eliminating any nonspecific binding.
Hope this will be helpful.
You can test to see if the problem is due to excessively high affinity of the protein for the resin by eluting the resin with EDTA, which will strip off the nickel. If the affinity of the protein for the nickel is the problem, the protein will elute with the EDTA. If the protein has precipitated on the column, EDTA will not elute it.
Convert the proteinyl histidines to the imidazolium B ion side chain form by dropping the pH to- ~5 in the presence of urea or guanidine. Protonated tau nitrogens so don't bind well and the chaotrope will solubilize the protein in case the acidity causes precipitation. You'll be able to keep Ni on your column and remove protein
You can also try to engineer a TEV site between your his-tag and protein. So after it gets bound to Ni-NTA, you can cleave the 6-his off the protein. Granted that your histidine amino acids of the protein will still have some affinity. You can then try eluting with imidazole.
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