Preparative IPG-IEF Apparatus

L. Ericsson (ericsson@u.washington.edu)
Mon, 19 Jan 1998 15:38:22 -0800 (PST)

Thanks to Hans-Werner Lahm and Gautam Sarath I know much more about the
problems involved with the operation of a preparative IPG-IEF apparatus,
but Kim Brown has raised the interesting question concerning the
solubility of the proteins. Certainly adding ampholytes to the buffer
defeats the purpose of the preparative method as mentioned by Lahm. In the
1997 Anal Biochem article by Pier Giorgio Righetti and Alessandra Bossi
they mention this problem. "Maintaining a protein soluble at its pI value
(i.e. in an ion-free environment) is the biggest challenge of all focusing
techniques, including IPGs. --- After much trial and error, we found that
addition of sugars, notably saccharose, sorbitol and to a lesser extent,
sorbose, greatly improved protein solubility in the pI proximity. The
improvement was dramatic if these sugars were admixed with 0.2 M taurine."
Then the article continues with conditions, concentrations, etc. followed
by an explanation of the phenomenon. "The results were explained with the
theory of Timasheff and Arakawa on stabilization of prtein structure by
solvents: sugars (at ca. 1M concentration) and zwitterions such as taurine
belong to class I stabilizers, characterized by negative binding to
proteins and by increasing the surface tension of water. As a result, the
protein is in a state of 'superhydration,' which might prevent binding to
Immobilines in the gel matrix and might alter some pKs on the protein
surface."

Has any ABRFer tried adding sugars and taurine to keep the isolated
proteins in solution? These are certainly much easier to remove from the
protein than ampholytes.

-Lowell H. Ericsson, Dept. of Biochemistry, U. of Washington, Seattle, WA