Nickel affinity purification

mnr mnr via methods%40net.bio.net (by mnr475 from gmail.com)
Fri Jun 3 05:57:40 EST 2011


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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