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Re: [ccp4bb] problem on protein precipitation
 

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CCP4bb <-- 2008 <-- January 2008 <-- 24 January 2008
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Date: 2008-01-24


Subject: Re: problem on protein precipitation
From: M T michelthe {- at -} GMAIL {- dot -} COM
Date: 2008-01-24
2008/1/23, Zheng, Lei :
>
> Hi ccp4ers,
>
>
>
> Sorry for this out-topic question:
>
> Recently we have a membrane protein expressed, after solubilized with
> detergent and purified from IMAC, the protein looks beautiful in SEC.
> However, it completely precipitates after the 2-3 days storage in 4 degree.
> We supplement 2 mM DTT in the new elute from IMAC, the protein looks happy
> during weeks at 4 degree. However, it starts to form an invisible aggregate
> (verified from SEC) during the protein concentration by Centricon. I know
> this is not uncommon problem for both soluble and membrane proteins and
> wonder if anyone has any tip and experience to overcome this problem.
>
> The protein pI is 8.6, buffer used is pH 7.6. Glycerol is always present
> during the purification. We do have high salt (500mM) in the buffer.
>
>
>
> Thank you for you input in advance,
>
> Lei
>
>
>
Hi Lei,

Maybe the detergent is not optimal for stabilization of your protein... You
can try an other detergent (longer, shorter, charged or not) or optimize
detergent concentration You can lost your detergent during concentration
(try a smaller cut off). An other consideration is on your storage buffer,
try other pH, other salts, other salts concentrations. Try to add a ligand
of your protein if it's possible. Try to cleave your his tag immediately
after purification (sometimes his tag cause precipitation). Do you know
something about possible lipidic cofactor? It can be crucial for protein
stability.
Each protein need its own optimized purification recipe and it's
particularly true for membrane protein.

Michel.
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