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:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger 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 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: rrowl...@colgate.edu 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 >
