On 14 Mar 97 at 9:48, Matteo Antorini
<matteo.antorini at bc.biol.ethz.ch> wrote:
>I need to isolate different isoenzymes, which are of similar isoelectric
>point. I tried to do it by preparative isoelectric focusing, but I can't
>get satisfactory results suitable for crystallographic studies.
>>I know of two products that might interest me: chromatofocusing columns
>and Isoprime (Multi-chambered Electrofocusing unit), both from
>Pharmacia.
>>Any experience report [snip] will be appreciated.
Hi Matteo,
I used Pharmacia's PBE94 chromatofocusing matrix and Polybuffer 96 for
a purification step in the purification of a bacterial amylase. My
experience tells me that for the separation of proteins of similar pI
it may not be totally satisfactory. While the protein I worked with
came off the column at exactly the same pH each time (which you would
expect) there was always a certain amount of 'tailing' so that some
active protein (<5%) would still come off when the pH gradient had
reached, say, 0.2 units below the enzyme's pI. If your isoenzyme's
have pI's that close together this might rule out the system. On the
other hand, I was pretty much packing alot of protein on the column.
I never got the kind of resolution claimed possible by Pharmacia so
maybe I was doing something wrong.
If you only need a few mg's recovery each run then low loadings WILL
improve resolution. The system's good points are that it is very
robust and provides perfectly reproducible results. On the down side,
the specialised buffers (Polybuffer 96 and 74) are expensive (for our
budget anyway) and the slow flow rates required for good resolution
means a single equilibration/separation run/cleaning cycle can take a couple
of days!
I am not familiar at all with the Isoprime system so I can't comment.
I can, however, point you in the direction of the Prep Cell from
Biorad. This system separates proteins on the basis of both their
molecular weight AND pI by 'continuous elution electrophoresis'.
Basically, the sample is loaded onto the top of a cylindrical
polyacrylamide gel (either native or SDS) and runs vertically down
the gel until its components run off the end where they are caught in
a constant flow of elution buffer which carries it off to a standard
fraction collector. The buffers used in the upper and lower resevoirs
as well as those used in preparing the stacking and resolving gels
can be varied to provide optimum separation.
This system has been used successfully in our labs for the
preparative separation of isoenzymes. It would, of course require an
investment from you in the equipment, and finding the right
combination of gel concentration and buffer combination can be
involved but it is worth checking out.
Hope this helps rather than confuses,
Cormac
P.S. Happy St. Patrick's Day to all bionet-heads!!
________________________________________________________________________
Cormac Shaw, BSc, |
Dept. of Industrial Microbiology, | e-mail: Cormac.Shaw at ucd.ie
University College Dublin, | phone: +353 (1) 706 1796
Dublin 4, Ireland | fax: +353 (1) 706 1183
------------------------------------------------------------------------
"Under the most rigorously controlled conditions of pressure,
temperature, volume, humidity, nutrients and other variables,
the organism will do as it pleases."
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