At 10:38 PM 7/22/97 +0100, John Sinclair <jcs at ermine.ox.ac.uk>
wrote:
>>Has anyone any experience, good or bad, of the BioRad Rotofor
in
>preparative IEF? Is it value for money in terms of ease of use
over more
>"traditional" methods (I've found a few in literature searches,
but all
>of them seem like they might take a while to get up and
working). Or
>perhaps someone could recommend a simple protocol that I might
be able to
>try? My protein is clean as judged by denaturing SDS-PAGE but
I'd like to
>make sure it's truely homogeneous. I'm hoping to clean up
milligram
>quantities.
>>Thanks in advance for any advice!
>>John.
>
We used it for a short time. Major problem is that
precipitating proteins tend to clog the
membranes separating the different compartments. Also, there
are only 20 compartments,
and emptying them was a little problematic. We now use the
Rainin RF3
preparative IEF instrument, which is the current cutting edge.
The sample circulates
through the system, with only about 10% of the sample in the
focusing cell at one time.
As it leaves the focusing cell, it is divided into 30 separate
channels, which go through
a cooling area and a bubble trap before coming back to the
focusing cell. Precipitated
protein simply circulates through the system (if focuses too)
and doesn't interfere with sample
focusing. The movement through the focusing cell is by laminar
flow, which preserves the
pH gradient and the separation of the sample. The system uses 2
different configurations,
one for 100 ml, the other for 500. The 100 ml system can handle
up to 100 mg of protein.
We use the system fairly early in our purification procedure, so
I don't know how it would
work in a later stage, but later is where IEF is traditionally
used. I think they also make a
smaller version of the instrument, which might work well at a
later stage.
The improvements in the technology are basically:
1. No barriers to be clogged by precipitated protein
2. Sample is separated as the run progresses, and doesn't have
the chance to remix.
3. 30 channels gives better fractionation.
I'm not affiliated with Rainin in any way, just an enthusiatic
customer. Good luck.
Phil Harrison
arsphys at cc.usu.edu
