Nickel affinity purification (mnr mnr)

Mark Nance via methods%40net.bio.net (by mrnance from umich.edu)
Fri Jun 3 13:26:42 EST 2011


Hi Matt,

With that much NaCl, you can probably omit the imidizole during the  
loading and wash steps.  One of my proteins requires 500 mM NaCl, and  
even though it has a 10-histidine tag it elutes at (or before) 50 mM  
imidizole.  If you still have your non-bound protein, you can dialyze/ 
dilute out the imidizole and run it back over the column.

- Mark



> Message: 1
> Date: Fri, 3 Jun 2011 11:57:40 +0100
> From: mnr mnr <mnr475 from gmail.com>
> Subject: Nickel affinity purification
> To: methods from magpie.bio.indiana.edu
> Message-ID: <BANLkTik9-6G30REknHBcWK=cgp-8LoMLkQ from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hi.
>
> My protein contains 6 histidine residues for His tag purification.  
> Less than
> a mg of protein is binding whereas therest about 10 mg don't. Both  
> bound and
> unbounds have his tag on western blot.
>
> I am using 10 ml of nickel sepharose from GE in 50 mM Tris pH 8, 500  
> mM NaCl
> and 10 mM imidazole in sample and equilibrate buffer. Protein elutes  
> at 200
> mM imidazole. Is there any way to increase the affinity of protein  
> binding?
>
> Thank you.
>
> Matt



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