Re: zinc finger protein purification issue

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Re: zinc finger protein purification issue

Dr Engelbert Buxbaum
Am 15.01.2010, 02:33 Uhr, schrieb 김종문 <[hidden email]>:

> I recently started to purify a protein containing zinc finger modif.
>
> the protein consist of flexible linker and his tag in the N term and C
> terminal of the mofit.
>
> the Zinc finger itself is consisted from three zinc finger motifs.
>
> It expressed well in the E.coli, and I was able to purify the protein,  
> but
> when I tried to concentrate it by using a membrane filter( amicon  
> centricon)
> the protein start to aggregate.

"flexible linker" may mean "intrinsically disordered", which would explain  
the behaviour. However, without knowning more about that domain, I can  
only guess.
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Re: zinc finger protein purification issue

wattne
It seems that your protein doesn't behave well at high concentrations.
Maybe it would help to have DTT or beta-Mercapto in your buffer. Or
you might try alkylating some Cystein residues to prevent di-sulfid
bridges? For concentration of proteins, I use Ammoniumsulphate
solution. You could try this and resuspend the precipitate in a buffer
containing DTT.

On Mon, 25 Jan 2010 17:39:20 -0400, "Dr Engelbert Buxbaum"
<[hidden email]> wrote:

>Am 15.01.2010, 02:33 Uhr, schrieb 김종문 <[hidden email]>:
>
>> I recently started to purify a protein containing zinc finger modif.
>>
>> the protein consist of flexible linker and his tag in the N term and C
>> terminal of the mofit.
>>
>> the Zinc finger itself is consisted from three zinc finger motifs.
>>
>> It expressed well in the E.coli, and I was able to purify the protein,  
>> but
>> when I tried to concentrate it by using a membrane filter( amicon  
>> centricon)
>> the protein start to aggregate.
>
>"flexible linker" may mean "intrinsically disordered", which would explain  
>the behaviour. However, without knowning more about that domain, I can  
>only guess.

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