However, these buffers generally absorb in the far UV (180-200nm) which is
the most interesting
part for discriminating the secondary structure distribution. Even if this
is not the primary interest, the 220nm
points one might want to use for a denaturation study can have quite some
background
contributions from the organic buffers.
Some alternative such as a thermofluor stability assay might not have the
problems
with organic buffers nor with the presence/absence of Mg++?
BR
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Roger
Rowlett
Sent: Wednesday, March 20, 2013 1:07 PM
To: [log in to unmask]
Subject: Re: [ccp4bb] suitable buffer for CD studies
I would use a low affinity metal ion binding buffer like MOPS, HEPES, or
MES. The "Good" buffers all have fairly low metal-ion affinity.
Phosphates will be problematic because of magnesium phosphate formation.
Cheers,
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [log in to unmask]
On 3/20/2013 2:59 AM, Harsh Bansia wrote:
>
> Sorry for a simple and non-CCP4 question.
>
> I have determined the structures of three different mutants of a
> thermostable protein by X-ray crystallography method. I feel that Mg2+
> has a role in protein stability.
>
> So I want to perform a thermal denaturation study by CD spectroscopy
> both in presence and absence of Mg2+ ion.
>
> In this regards, what should be the suitable buffer for CD studies?
> May I use PBS buffer ? Since phosphate sequester divalent cations like
> Mg2+. Is it advisable to use PBS buffer. If so, what is maximum
> concentration of Mg2+ ion that can be used say e.g. 5 mM? My protein
> was in Tris buffer and lyophilized and have theoretical pI =4.56 and
> maximum activity at pH 8.4.
>
>
> Thanking you in advances,
>
> harsh
>
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