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Purifying an ERM protein: is Ni-NTA compatable w/ 2M NaI?

Warren Gallin wgallin at gpu.srv.ualberta.ca
Wed Mar 31 23:02:08 EST 1999


In Article <jkurtis-3103992233450001 at dialin0716.upenn.edu>,
jkurtis at mail.med.upenn.edu (Jonathan Kurtis, MD/PhD) wrote:
 
>I'm purifying an actin-binding protein from Pichia Pastoris (an
>Ezrin-Radixin-Moesin family member). My 6xHis tagged protein of interest
>(~80 kDa) co-purifies on NiNTA resin with an ~50 kDa band in a ratio of
>85% protein of interest to 15% unwanted contaminant. This contaminant is
>not removed by subsequent anion exchange or HIC chromatography despite
>playing with detergents and ETOH and glycerol. It turns out that the 50
>kDa contaminant is probably a binding protein (EBP50) that associates
>strongly with ERM family members. In the literature, these two interacting
>proteins have been separated by an affinity matrix (immobilized ERM used
>to purify recombinant EBP50) with a 2M NaI elution.
>
>My question: Can I bind my yeast lysate onto Ni-NTA in the presence of 2M
>NaI?? This should allow only my protein of interest to bind with the EBP50
>contained in the flow-through. Has anyone tried this or similar high salt
>concentrations?? Anyone know what 2M NaI does to a protein??

NaI is chaotropic.  Although I couldn't say for certain that you can use
NaI, I routinely purify His-tagged proteins on nickel columns using
solutions with either 8M urea or 6M GuHCl, both strongly shaotropic agents. 
The problem with using NaI might be if the iodide reduced the metal ion (I
don't know if this will happen or not, just something that you might want to
watch out for).
    Anyway, if you bind your His-tagged protein in 6 M GuHCl and include 8M
urea in your wash and elution buffers, I would be surprised if your binding
protein came along for the ride.
Warren Gallin
Department of Biological Sciences
University of Alberta
Edmonton,  Alberta     T6G 2E9
Canada
wgallin at gpu.srv.ualberta.ca



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