Dear Atul,
for crystallisation, I would say the most suitable buffer is water.
If your protein concentrates well in pure water, why add anything else?
Of course, many proteins will not concentrate well in pure water, so
start adding buffer components until your protein is happy:
- buffer (HEPES, Tris) at 10 mM.
- a bit of salt
- ions your protein may need for stability
- etc.
The thermofluor (Analytical Biochemistry, Volume 357, Issue 2, 15
October 2006, Pages 289-298) assay can be good for this.
Mark J. van Raaij
Dpto de Bioquímica, Facultad de Farmacia
Universidad de Santiago
15782 Santiago de Compostela
Spain
researcherID: B-3678-2009
On 11 May 2009, at 07:48, atul kumar wrote:
i dont know how people decide that this buffer ph ,salt
concn,dtt,etc in perticular concm is suitable for protein to be in
folded state,i am doing crystallisation of few proteins but i dont
know how to select the parent buffer for the protein,is it CD or
crysatllisation trials that tells abt this
-Original Message-
From: CCP4 bulletin board on behalf of James Stroud
Sent: Mon 5/11/2009 8:59 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Manipulating electron density
On May 10, 2009, at 12:21 PM, Phoebe Rice wrote:
> Of COURSE the map will look lovely if you carve it off at 1.5A
> from your atoms. And your gels will look lovely too if you
> just touch them up with with some white-out and a sharpie.
> Do the honest thing and show the whole truth, using the
> z-clipping to get a comprehensible slab.
Maybe we should give Jason the benefit of the doubt that he wants to
present an honest representation of his density. Clipping off symmetry
related molecules is not the same as "carving" density from amorphous
blobs. The former is more akin to showing only part of a lane of a
gel, which is common practice even in prestigious journals. In fact,
my other monitor presently displays such a "carved" gel picture from a
recent Cell paper.
James
