Re: [ccp4bb] Solubilization buffer

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

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Subject: Re: Solubilization buffer
From: Dima Klenchin klenchin {- at -} FACSTAFF {- dot -} WISC {- dot -} EDU
Date: 2009-12-02

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Subject: Re: methews coefficient and validation of 60mer
From: Pankaj Chauhan pankajimtech {- at -} GMAIL {- dot -} COM
Date: 2009-12-02

Subject: Re: Solubilization buffer
From: "John A {- dot -} Newitt" newittja {- at -} GMAIL {- dot -} COM
Date: 2009-12-02

At 10:35 PM -0600 12/2/09, Dima Klenchin wrote:

>2. Chances are good that you can use as high temperature as you need
>to dissolve. I've come across protocols using >=65C in the presence
>of 100 mM bME to ensure complete unfolding of the polypeptide and
>reduction of disulfides (seems essential for refolding of some
>proteins and very detrimental for others).

I wouldn't recommend using hot 8 M urea to dissolve proteins that you
plan to re-fold for functional studies or crystallization since
heating the urea solution will produce a significant amount of
isocyanic acid which will react with primary amines in your protein
(including the N-terminus) and produce carbamylation modifications
(-NH2 to -NHCONH2). If it is that tough to dissolve your inclusion
body, I would use 6 M guanidine HCl (in ~200 mM Tris-HCl pH 8.5, 1 mM
EDTA, 20-100 mM DTT).

You can make 8M urea stocks by dissolving at room temperature (20-25
deg C) but you need to be patient (don't heat). Include an
amine-containing buffer like Tris in your final solubilization
solution (to help scavenge isocyanate), and don't use old urea stocks
since the dissolved urea will decompose into ammonia and isocyanate
with time.

- John
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CCP4bb navigation

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

Previous message:
Subject: Re: Solubilization buffer
From: Dima Klenchin klenchin {- at -} FACSTAFF {- dot -} WISC {- dot -} EDU
Date: 2009-12-02

Next message:
Subject: Re: methews coefficient and validation of 60mer
From: Pankaj Chauhan pankajimtech {- at -} GMAIL {- dot -} COM
Date: 2009-12-02