Re: [ccp4bb] Solubilization buffer

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Re: Solubilization buffer
From: Savvas Savvides savvas {- dot -} savvides {- at -} UGENT {- dot -} BE
Date: 2009-12-03

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From: MARTYN SYMMONS martainn_oshiomains {- at -} BTINTERNET {- dot -} COM
Date: 2009-12-03

Subject: Re: Solubilization buffer
From: natrajan natrajan {- at -} EMBL {- dot -} FR
Date: 2009-12-03

Hi Megha,

You could make your buffer solutions at room temperature, so
that the 8M urea dissolves completely. During solubilization of the
protein, keep the tube on a shaker or agitate the solution using a magnetic
strirrer. This will prevent any crystallization of the urea. In any case,
urea crystallizes very slowly, even at 4 degrees C, as already mentioned .

Another issue is the solubilization time. You've mentioned that you do it
only for 1 hour. In the proteins I have worked with, I have had to
solubilize overnight to get a reasonable yield. Of course this could be
different for each protein.

Hope this helps

Ganesh

On Wed, 2 Dec 2009
20:16:09 -0800, megha goyal wrote: Hi all, We use 8M urea
solubilization buffer for our protein in inclusion bodies and recommended
temperature is 10-15Âº C. but in 8M conc the urea does not dissolve and is
in crystalline form only, will it have any effect on solubilzation
efficiency. Our solubilization time is 1 Hr and after that we centrifuge
and use the supernatant for refolding via dialysis. however the pellet
after centrifugation of solubilzation show presence of our protein on sds
page analysis. what should we do so that the process of solubilization is
complete and our protein is not lost in pellet. thanks in anticipation.
meg

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Solubilization buffer
From: Savvas Savvides savvas {- dot -} savvides {- at -} UGENT {- dot -} BE
Date: 2009-12-03

Next message:
Subject: Re: Refining residues as rigid bodies
From: MARTYN SYMMONS martainn_oshiomains {- at -} BTINTERNET {- dot -} COM
Date: 2009-12-03