From: John Correia <jcorreia@biochem.umsmed.edu>
  To  : jphilo@mailway.com
  Date: Sat, 28 Jul 2001 14:27:54 -0500

RE: small peptides, offsets

John

With regard to large offsets - it is far to say that interference offsets are speed dependent, strain in the windows is the usual excuse, but aside from your experience I am not aware of much discussion or evidence for absorbance offsets being or not being speed dependent.  I'll keep it in mind in the future.

Clearly large offsets can be due to dirty optics, presumably something we can fix, but I think in my case its the reductant.  All my recent work on Smads is in 2 mM TCEP, and while the reports are that it doesn't absorb, I have observed slow drifts in offset during a run, although its much worse if you don't blank with TCEP too.  There are reports that protein plus TCEP causes the drift, but I am not aware of a chemical mechanism being reported.


>With regard to what is a reasonable value for an absorbance offset, I must
>say that IMHO unless you have reductants in there, if you get offsets of
>0.05 AU then there is something wrong with your samples or your instrument.
>Usually a large offset means there is some contaminating small molecule
>contributing to the absorbance, and in such cases you may see a strong
>correlation of the offset with the protein concentration. One source of such
>contaminants are those *!*#!! color-coded plastic tubes some labs are found
>of (throw them out!).