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Re: [ccp4bb] protein degradation during concentration for crystallization trials
- Protein crystallography

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999
Previous message:
Subject: Re: question about the zinc binding protein
From: Peter Hsu hsuuper {- at -} U {- dot -} WASHINGTON {- dot -} EDU
Date: 2010-04-09		Next message:
Subject: Fw: P-CUBE Workshop & User Meeting, September 8-9, 2010]
From: Andrew McCarthy andrewmc {- at -} EMBL {- dot -} FR
Date: 2010-04-09


Subject: Re: protein degradation during concentration for crystallization trials
From: Peter Hsu hsuuper {- at -} U {- dot -} WASHINGTON {- dot -} EDU
Date: 2010-04-09

Hi,

I've not tried this on column cleavage before, but have you tried first purifying the protein. cleaving the tag off the column and rerunning it through the column to capture the tag and washing off the protein?

Also, with concentration. Going from a 50 mM buffer, and .3M salt, down to nearly water may not be great thing to do. Can your protein concentrate in your purification buffer to about .1-.2mM? If it can, I highly suggest trying to use the dialysis buttons from Hampton and screen a variety of different pHs, salts and additives to see what your protein can tolerate, before committing an entire purification prep to concentration.

I've also noticed if you're using the centricon concentrators, make sure you take it out every few minutes and pipette it the solution a bit. These concentrators tend to create a concentration gradient, making the local concentration of protein very high in bottom of the concentrator, often times resulting in ppt.

Hope it helps

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999
Previous message:
Subject: Re: question about the zinc binding protein
From: Peter Hsu hsuuper {- at -} U {- dot -} WASHINGTON {- dot -} EDU
Date: 2010-04-09		Next message:
Subject: Fw: P-CUBE Workshop & User Meeting, September 8-9, 2010]
From: Andrew McCarthy andrewmc {- at -} EMBL {- dot -} FR
Date: 2010-04-09
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