Dear all,
I am trying to elute my protein of interest (POI) from Ni-NTA column without using imidazole, since this is incompatible with Cation-Exchange-Chromatography, which will be the subsequent step in my purification strategy.
I thought about using pH elution. Has anyone experience with that? Will it strip the metal from the column? If yes, at which pH? Is a gradient or a step elution recommendable? Can pH modulation be used as efficient washing step?
I planned to wash with a buffer of pH 6 and then elute with a buffer of pH 4.5 - will this strip metal from the complex? I don't want to have nickel in the eluate.
Thanks for any help!
Peter
Hi there,
Nickel binding to NTA is stable in a large range of pHs according to manufacturer's leaflet as pH elution is performed at pH4.5 in some protocols.
If the purpose of pH elution is to avoid imidazole for IEC, you may still consider imidazole elution and get rid of it through dialysis or gel filtration before the IEC step.
IMAC has to be at a pH above the pKa of a His, usually well above pH 6, (~7.5) and the pH shift simply makes the His uncharged and it detaches from the Ni2+, in my understanding. You will always have (a little) Ni2+ in the eluent, but its usually not an issue. EDTA elution would strip the metal, but low pH elution, if tolerated by your POI, should be AOK.
Yes, you can elute it by decreasing pH below 6.0, but practcally your protein may not get eluted completely you need to add some NaCl to get complete elutioin. If you want to directly load the IMAC elution on cation exchanger you can try the following.
IMAC elution can be carried out by decreasing pH, increasing imidazole counter ions or by stipping the metal ions with EDTA.
Elute at lower pH if it eluted at pH 4.0 then it is ok you can directly bind it to cation exchanger with the same pH, if not eluted you need to add some 200mM NaCl for elution this is need to dilute until it comes to below 4ms/cm^2.
Eluting with EDTA directly strip off the metal along with protein, the amount of Ni ions may the proble to loading on ion exchanger.
Eluting with imidazole also give the same problem this can be loaded by diluted or buffer exchanged to load on ion exchanger.
we all wants to reduce the steps in downstram, but in this case if it is eluted at lower pH it is ok, but if you choose the other options you may need to include a buffer exchange step or you may need to dilute the sample or may be HIC can be used to load directly with salts but purity has to compare with ion echanger.
Thanks for all the input and suggestions! I will then load the sample in pH 8 with 0.2 M KCl and wash with a gradient from pH 6 to 5. Then elution at pH 4.
Collect the wash! (I suspect you may see some elution at pH 5) Good luck!
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