Re: HPLC preparative IEC columns
Gautam Sarath (gsarath@unlinfo.unl.edu)
Fri, 22 May 1998 13:14:44 -0500 (CDT)
Dear Corrado: Our experience with POROS have been mixed. It works well
for some proteins and not at all for others. It is however the first
column to try for recombinant proteins. We purify all of our synthetic
peptides on POROS columns, and are gearing up to do the same with
recombinant proteins. I have no experience with the Prep columns, but the
analytical ones give very reproducible chromatograms, esp when attached to
the BioCAD Workstation. If you expect a lot of contaminants in the crude
extract, it should be possible to first run the sample on a Pharmacia High
Flow Phenylsepharose (hig sub) packing at about 7 ml min to achieve
separation of the protein from other materials. This can be suitably
diluted and directly loaded onto the Cation column. In our hands the Poros
HS-M packing behaves somewhat between the CM-type and the SP-type of
exchangers. The purification is good when there are less than 10 other
proteins, we have never gotten good good separation in solutions containing
more components, unless they show great differences in their binding
behavior ie., it is very difficult to resolve shoulders on the Poros
resins. We have also occassionally lost over 50 % of the activity on the
Poros packing as compared to the old fashioned Cellulose-based IECs. good
luck, gautam
Gautam Sarath
N-226, Beadle Center
Protein Core Facility - Center for Biotechnology &
Department of Biochemistry
University of Nebraska-Lincoln
Lincoln, NE 68588-0664
Phone: 402-472-2928
FAX: 402-472-7842
http://www.biotech.unl.edu/Proteins/index.html