Hi,
One has to bear in mind that adsorption at high pH (above 6.5 is already
high for Imac; pKa of exposed His is 6.2) leads to stronger interactions
which, in turn, require stronger eluting conditions, e.g. > 200 mM
imidazole (pKa ~ 7.0).
I suggest lowering the pH of the adsortion buffer, e.g. 6.5 or even 6.0
(try and see) so that you dont need to go beyond 100 mM
imidazole in your elution buffer (as mentioned earlier, you must ajust
the pH of the latter as required with extra HCl).
Best,
Nadir Mrabet
--
Pr. Nadir T. Mrabet
Cellular & Molecular Biochemistry
INSERM U-724
Nancy University, School of Medicine
9, 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
E-mail: [EMAIL PROTECTED]
Ronnie Berntsson wrote:
Hi,
In addition to the tips already suggested, you could also try to elute
your protein with histidine.. I've got a protein that crashes out with
imidazole as well, and I have succesfully used 200mM histidine for
elution (using either KPi or MES buffer in my case).
Cheers,
Ronnie
On Feb 15, 2008, at 5:47 PM, Juliana Barbosa Coitinho wrote:
Dear all,
I am with the same problem of Jacob.
My protein is precipitating, especially when I 'freeze it'. I am
using a His Trap HP column coupled with a desalting column. Already
tried to elute with gradient of imidazole (that allowed an elution
with less amount of imidazole), but even then, the protein still
crashes-out.
I am trying to use the protein immediately after purified it, but
this is not always possible and I am losing much protein with that.
I will try to use the tips that have already been said.
But if someone can help me more, I will thank!!!
Thanks a lot!
Juliana
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Ronnie Berntsson
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Ph.D. Student
Department of Biochemistry
Groningen Biomolecular Sciences and Biotechnology Institute
& Zernike Institute for Advanced Materials
University of Groningen
Nijenborgh 4, 9747 AG
Groningen, The Netherlands
telephone: +31 50 363 4195
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e-mail: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>
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