Re: Protein Purification using isoelectric focusing with

Gautam Sarath (gsarath@unlinfo.unl.edu)
Mon, 19 Jan 1998 08:56:29 -0600 (CST)

In my hands, I've had a fair bit of trouble with the rotophor system,
primarily with protein precipitating on the membranes and the need to
repeat the separations at least a couple of times. I've also tried the
BioRAD rotolytes, (non-ampholines) and did not observe much success with
the two trials I did. I know that the rotophor cell works quite well for a
few select systems, but if recovery and yields are critical, it is probably
not the bbest system when protein is limiting. We have used the BioRAD
prepcell and the rotophor for separation of recombinant protein, where we
could take losses, it was not much different than a series of columns,
considering that the yields on the rotophor system were low. We have also
failed to separate isozymes differing by a few tenths of a pH unit on the
Rotophor, but get excellent resolution on standard vertical IEF gels.
However, I have seen some excellent data on the separation of fungal
peptidases by the Rotophor. Thus it seems to be very system specific.
Also, it has the lure of the bright lights of a Casino, but the chances of
getting the Jackpot are difficult. gautam

>At a recent conference this year Pier Giorgio Righetti described his
>immobilized pH gradients (IPG) apparatus with zwitterionic membranes
>creating "isoelectric traps" for individual proteins thus allowing for
>continuous purification of proteins on a pilot plant scale basis. The
>apparatus is described in an article in Anal. Biochem. Vol.247 pg1-10
>(1997).
>
>With this apparatus one can realize the advantages of isoelectric focusing
>without the problems involved with separating the protein from acrylamide
>or ampholytes.
>
>Several similar apparatuses (Rotofor, Elphor, etc) to the Righetti
>apparatus sold by Pharmacia-Hoefer have been on the market the last few
>years but I seldom see a publication in which these instruments have been
>used. What experience have the ABRFers had with these instruments? They
>appear to be just the thing for purification of proteins for NMR, x-Ray,
>and MS studies. No acrylamide or ampholytes to worry about, or is their a
>problem?
>
>-Lowell H. Ericsson, Dept. of Biochem., U. of Washington, Seattle, WA

Gautam Sarath
N-226, Beadle center
Protein Core Facility
Department of Biochemistry
University of Nebraska-Lincoln
Lincoln, NE 68588-0664
Phone: 402-472-2928
FAX: 402-472-7842