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
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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
>
> ----------
> 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
>
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>
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