Respected All,
Thanks for your valuable suggestions and inputs.
with regards,
Harsh
On Thu, Mar 21, 2013 at 7:38 AM, Bosch, Juergen jubo...@jhsph.edu wrote:
Yep,
mostly you should stay away from Tris as this is the worst buffer system
when playing with temperature changes. Tris for
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
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
Rowlett
Sent: Wednesday, March 20, 2013 1:07 PM
To: CCP4BB@JISCMAIL.AC.UK
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
Harsh,
This article describes common buffers for CD on page 2:
http://www.ncbi.nlm.nih.gov/pubmed/17406547
Or this article on page 8:
http://www.ncbi.nlm.nih.gov/pubmed/16027053
It seems like phosphate is the best, because it has low absorption at
180-200nm region. From organic buffers
Tris-sulfate might be acceptable for studies in the presence of
magnesium ion. Na-MES is tolerable at low concentrations and shorter
path lengths if a cutoff of 190-200 nm or so is acceptable. Chloride is
indeed problematic in the far UV.
___
Roger S.
I have found it is best to test the absorption of your buffer in the
wavelength range you are interested in.
If you are going to do a temperature study with CD perhaps at 222 nm, then
test your buffer there with your UV spec.
You want to have little or no absorption. Or do the range 200-270 nm
Sent from my iPad
On 20/03/2013, at 7:59 PM, Harsh Bansia spideysp...@gmail.com 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
Hi Harsh
Something like sodium borate at pH 9.0 could be an alternative to phosphate
buffers. If you are looking at thermal unfolding above 220nm, then the choice
of buffer is less critical as many buffers and additives are problematic only
below 200nm.
If your samples require high salt
Why not thermal denaturation in the presence of Sypro Orange and a realtime PCR
machine ?
Crowther et al. Use of thermal melt curves to assess the quality of enzyme
preparations. Anal Biochem (2010) vol. 399 (2) pp. 268-275
Or (shameless advertisement):
Hain et al. Structural characterization
One of the other things you need to be concerned about with thermal melts
is the change in buffer pKa as temperature varies (I seem to remember this
being called the beta factor). Phosphate is used for CD melts regularly
because its pKa is fairly invariant with temperature. (A good reference is
Yep,
mostly you should stay away from Tris as this is the worst buffer system when
playing with temperature changes. Tris for example has a ∆pKa/10˚C -0.31
Good, N.E. (1986) Biochemistry 5, 467
Jürgen
P.S. @Matthew, was this what you meant by the Good buffers often not ? or
just a
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