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 and choose a buffer that does not absorb in that range.
For example we thought Bicine pH 8.5 with BME would be ok for CD studies. Bicine pH 7 did not absorb at 222 nm, but 8.5 gave a high background. bME really absorbs We ended up using PBS with a smidgeon of BME. Also you should use a protein concentration that gives you an absorption of 1.0 at the wavelength of interest This is crucial to keep noise out of your data. Also the CD dynod voltage should be monitored and keep it below 400 for protein studies Here is an excellent reference: Kelly, Ness and Price (2005) "How to study proteins using circular dichroism" Biochemica et Biophysica Acta *1751*, 119-139 Good Luck, Gloria On Wed, Mar 20, 2013 at 1:59 AM, 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 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 >
