______________________________ Reply Separator _________________________________
Subject: RE: Protein Purification using isoelectric focusing with zwi
Author: "Hans-Werner Lahm +41 (61) 688-3912" <HANS-W.LAHM@roche.com> at
Internet
Date: 1/19/98 5:07 AM
Our protein crystallography group got a preparative IPG-IEF apparatus from G.
Righetti's lab about 7 years ago. Here are our observations:
-The technology is very labor intensive, as the analytical IPG strips and the
preparative IPG membranes have to be prepared under the same conditions with
the same set of immobilines (Fluka).
-IPG-based IEF has a much lower conductivity when compared to conventional IEF
based on ampholines. Practically, this means that you have to add ampholines,
salts,.. to your buffers, thus negating some of the advantages using
preparative IPGs in the first place.
-The preparative method did not work on most of the proteins we were
interested in. The proteins precipitated on the membranes thereby clogging
their pores.
However, we found IPG-IEF such an excellent method for analytical IEF that we
converted all our 2D gel methods to IPGs. Since about 5 years precast
analytical IPG strips are available commercially (Pharmacia).
Hans-Werner Lahm
hans-w.lahm@roche.com