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Difficulty eluting His-tag Zn metalloprotease - (Feb/16/2015 )

Hello everyone,


I was recently attempting to purify a 6x His tag recombinant fungal effector which had a natural Zn-binding motif. I tried eluting in high concentrations of imidazole, even resorting to adding EDTA. I got up to 250mM EDTA and 1M imidazole with 0.25% tween 20 but still the protein remained on the Ni-NTA beads.. It took heating the beads to 92degrees with 10% SDS to disrupt the interactions.


Is there any explanation for this unusually high affinity for Ni-NTA? Any suggestions of what I could do now? Could I change the beads to Ni-IDA, as I've heard they have a lower affinity for Histidine? 


Many thanks,





i suspect that the zinc in your protein is binding directly to the nta.


you can try replacing the nickel on the matrix with zinc so that the zinc in the protein may not preferentially bind to the nta.


here is the ge life sciences handbook webpage (you can download the handbook for purifying recombinant proteins, amongst other useful handbooks).