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 <[email protected]>
Subject: Nickel affinity purification
To: [email protected]
Message-ID: <[email protected]>
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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