> Dear ABRFers,
>
> We are interested in purchasing a system for running 2D gel
> electrophoresis. Can anybody tell us what is their experience with
> Pharmacia's Multiphor II System and their Immobiline DryStrips? Can samples
> in urea be loaded on the strips? Also, has anybody worked with their newest
> IPG4 system for 2D gel electrophoresis?
> We were also thinking of using BioRad' system, but as of now, they don't
> sell either the IPG strips nor the apparatus for running them.
>
> Thanks.
>
> Dobrin Nedelkov
Dear Dobin,
I have been using the Pharmacia's Multiphor II System for 3 years with quite a
lot of success so far, using both the conventional cup loading and the "in gel
sample rehydration method". The Immobiline strips, especially the 4-7 Linear
one, offers good to exceptional pI separations and I have been very satisfied
with them, in quality and ease of use. The batch to batch reproducibility is for
that matter not as good and the Immobiline strips can vary in length up to one
cm in the worst case. Recently, Pharmacia has also released a serie of
ampholines that should enable you to pour your own strips, especially useful if
you want to pursue basic proteins (see also a recent publication from Angelika
Goerg about handling basic proteins). The one caveat I would like to add is that
I am not thrilled by the protocols given with the apparatus. I had often
problems and ended up to modify all of them. In particular, the protocol
provided with the "in gel sample rehydratation method" was particularly obscure
and I had to sacrify a couple of strips in order to find the right technique.
Now, to answer some of your questions in particular:
"Can samples in urea be loaded on the strips?" Of course! Actually, urea is a
key element to prevent proteins to precipitate during the focusing time.
"Also, has anybody worked with their newest IPG4 system for 2D gel
electrophoresis". Personally, I have not, but I would be interested in hearing
what others have experienced with it.
A last comment: I would not recommend using the flat bed electrophoresis system
of Pharmacia for the second dimension due to its low throughput (You can run up
to 12 first dimensions simultaneously, but only 2 second dimension). I recommend
either the IsolDalt system from Hoefer (10 gels simultaneously, very
reproducible conditions from run to run, however quite pricy and not that easy
to handle), the Investigator from ESA (5 gels simultaneously, usually quite
reproducible) or even a couple of the BioRad systems (You will have to cut the
extremities of the strips if you use the 18 cm version, though).
Finally, remember that the right equipment is only half the work. 2-DE is not a
simple technique, with many steps and many potentilal pitfalls. Good gels are
usually the results of hard work and, sometimes, quite a lot of frustration.
Hope that it helps.
Axel
--------------------------------------------------------------------------------
Axel Ducret, Ph.D.
Senior Research Biologist
Merck-Frosst Canada Inc.
Dept. Biochemistry and Molecular Biology
P.O. Box 1005
Pointe-Claire-Dorval PQ H9R 4P8
Canada
tel. + (514) 428-3428
fax + (514) 428-4900