You might be giving too much importance to the THEORETICAL pI of the protein. If it's supposed to be well charged at pH 7,4 (only a titration curve, and not simply knowing the pI will tell you this) and it's still precipitating, the problem might be due to a bad fold, for instance, or to the lack of salt ...
I've heard people saying that proteins with high pIs are more difficult to work with, but to be honest I don't know where that fear comes from.
Carlos
Em 02/03/2012, ās 05:55, Artem Evdokimov escreveu:
> This pH is generally incompatible with Ni IMAC, sorry :) If you have a
> high pI your best bet is to employ ion exchange as primary capture,
> specifically SP resin or if you're really lucky - CM resin. There are
> only relatively few proteins in E. coli that bind to CM resin at pH 5
> and virtually none (one-three) that will bind at pH 8. If your protein
> still binds, then you're good to go.
>
> Artem
>
> On Thu, Mar 1, 2012 at 10:49 PM, anita p <[log in to unmask]> wrote:
>> Hi all,
>> Has anyone used sodium acetate buffer pH (4-5) for purifying histag protein
>> on Histrap column (AKTA) followed by SEC?
>> My protein has a pI of 9. I tried pH7.4 but it has precipitation problems.
>> While doing buffer screening using 24 well hanging drop I found that lower
>> pI onces are clear, so just thinking can I use Na acetate at pH 5 for whole
>> purification???
>>
>>
>> Thanks in advance
>> Anita
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