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Hi Capricy,
How did you decide to use anion exchange rather than cation exchange?
What if your protein has negative-cluster patches that prevent it from
interacting effectively with your negatively-charged column?
What if only those patches are available on the surface when the protein
is at high concentration (concentration driven assembly)?
Why not try hydrophobic chromatography (assuming your protein bears few
charges, non accessible charges, or paired charges only)?
Also, you should have an idea of the pI of your protein, at least from
its sequence. This would allow you to adjust the pH of your buffer
to promote adsorption.
My suggestion would be that you have a look at the free (excellent) pdf
booklets available from Pharmacia (GE Healthcare/Amersham/Pharmacia) at
http://www1.amershambiosciences.com/aptrix/upp00919.nsf/Content/LabSep_HomePage.
Hope it help(ed)s.
nadir
======================================================
Pr. Nadir T. Mrabet
Cellular & Molecular Biochemistry
INSERM U724
UHP - Nancy I, School of Medicine
Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax: +33 (0)3.83.68.32.79
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capricy gao wrote:
Hi
My protein of around 15mg/ml (0.3mM) was loaded to anion exchange
column with 25mM Tris.HCl, pH8.0, but it came out before the gradient
started, never sticking to the column. After I did the dilution by 10
to 15 fold, it showed to bind to the column.Anybody has the similar
experience and share some explanations?
Thanks,
Capricy
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