To separate protein from Ni+2/EDTA

D.K. DK at no.valid.address
Tue Jan 14 21:40:19 EST 2003


In article <AQ2V9.1555$i73.314446 at twister.neo.rr.com>, "Artem Evdokimov" <AEVDOKIMOZ at cinci.rr.com> wrote:
>"Debashis Mukhopadhyay" <debmukh at scripps.edu> wrote in message
>news:Pine.SGI.4.20.0301141518540.199543-100000 at signal.scripps.edu...
>> Hi,
>>
>> I am using Ni affinity column to isolate my protein which came out
>> successfully with Imidazole. However, I found a lot (~100 mg) of protein
>> still sticking to the column and I eluted them out by stripping off the
>> Ni+2 from the column using EDTA. So, this solution now contains my protein
>> + (may be some contaminant) + Ni and EDTA and is colored blue (as
>> expected). At this point I wonder if there is any way / standard practice
>> to separate the protein from Ni+2, so that I can re-utilize it.
>>
>> Any help would be appreciated.
>>
>> Regards,
>> Deb
>
>It's not very likely that the protein would be OK. In my experience, strange
>things happen to proteins exposed to soluble nickel salts, and Ni tends to
>persist even after thorough cleanup. 

Sounds strange. One would think that EDTA is in large excess and 
since it binds Ni with Ka > 10e13 there would be no Ni bound to 
protein. 

>You can try, however - simple desalting
>(size-exclusion) should do the job. After desalting, remaining nickel can be
>sometimes scavenged using chelating resins.

If the volume is large, simple dialysis should do the job too. 
To be on a safe side, I'd dialyse first against 1 mM EDTA 
(with a change) and only after that in whatever other buffer 
that suits me. 

DK



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