[RASMB] DDT, detergents, and reference intensities
John Correia
jcorreia at biochem.umsmed.edu
Mon May 12 17:31:01 PDT 2003
To follow up Les' DDT calculation, one cannot assume that the sample and reference will oxidize at the same rate & to the same extent. Thus, during a long run you still may develop mismatch - I think Tom Laue has reported this for DTT suggesting blowing out the cell with N2 before cell filling, and we have seen similar baseline drift with TCEP, which at least has a smaller extinction coefficient.
I recall doing a sed vel run on TMVP with PO4 and interference optics 25 years ago - I mistakenly diluted a pH 7 sample with a pH 6.5 buffer, same PO4 concentration otherwise. The pattern was all monobasic / dibasic mismatch and uninterpretable. The power of dn/dc is it is very sensitive to any concentration gradient.. The danger is it is very sensitive to any concentration gradient..
-------------------------------------------------------------------
Dr. John J. "Jack" Correia
Department of Biochemistry
University of Mississippi Medical Center
2500 North State Street
Jackson, MS 39216
(601) 984-1522
fax (601) 984-1501
email address: jcorreia at biochem.umsmed.edu
homepage location: http://biochemistry.umc.edu/correia.html
dept homepage location: http://biochemistry.umc.edu/
-------------------------------------------------------------------
>>> <HOLLADAYL at aol.com> 05/12/03 04:08PM >>>
Hi all;
the disulfide chromophore has an extinction coeifficient of around 110 at
280. Now if one has 200 mM DDT, and 10% is oxidized, then one has a
background buffer absorbance of 2.2 at 280.
My counsel is to always run a bench top absorbance scan of both buffer and
sample if it is a sample given to you. I think all of us have horror stories
of hysterical samples.
As for detergents, I've seen problems with sed eq data at higher speeds when
the micelles redistributed enough to throw the light beam off center towards
the bottom.
There is no substitute for looking at the intensity of the reference sector
for equilibrium absrobance scans. This will tell you the story. I take
credit for this being in the Beckman acquisition software.
best regards
Les Holladay
More information about the RASMB
mailing list