[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..





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 Dr. John J. "Jack" Correia
 Department of Biochemistry
 University of Mississippi Medical Center
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>>> <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





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