Load your sample, wash with plenty of buffer and then elute with the same buffer containing let's say 10-100 mM EDTA (the higher concentration, the sharper peak you should get). After that you'll need to dialyze of concentrate and wash on Amicon to remove the EDTA-Ni.
What should be the components of my washing and elution buffer ? and what should be their relevant pH?
Hi zaheer,
Poly-His tags bind best to IMAC resins in near-neutral buffer conditions (physiologic pH and ionic strength). A typical binding/wash buffer consists of Tris-buffer saline (TBS) pH 7.2, containing 10-25mM imidazole. The low-concentration of imidazole helps to prevent nonspecific binding of endogenous proteins that have histidine clusters.
High concentrations of salt and certain denaturants (e.g., chaotropes such as 8M urea) are compatible, so purification from samples in various starting buffers is possible.
Elution of His-tagged protein from an IMAC column is accomplished with a high concentration of imidazole (more than 200mM) with other similar buffer condition as binding or wash buffers.,
Thanks all. So if i make my elution buffer with 50mM EDTA, 500mM imidazole (typical component of elution buffer ) , 0.25M NACL and 10mM Tris, Do you think it would work for me? and Should i need to adjust the pH to 8.0 upon the addition of EDTA or there is no need to do that? Please share your views. Thanks
So you have never done the binding to the Nickel column before? I thought you did and just want to change the elution mode.
Anyway, there is no need to add both EDTA and imidazol, use either of that.
10 mM Tris is quite low, it won't have sufficient buffering capacity and since you do not have to worry about ionic strength (as in case of ionex), you can use 50-100 mM concentration.
The EDTA is best prepared as stock with pH 8.0 anyway, so if you add it to buffer with pH 8.0, it should be OK, but I'd check it at least the first time just to make sure, because you'll have quite lots of the EDTA.
Thanks all for the useful suggestion and providing me the best of your knowledge. Thanks again. The problem has been troubleshooted.
Elution was done using 50mM EDTA with 500mM imidazole.
500mM imidazole gave weak elution
50mM EDTA alone didn't give any elution at all.
Thanks
Regards!
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