In article <kennedym-0607951104200001 at kennedymi.mayo.edu>, kennedym at mayo.edu
says...
>>Hello,
> Has anyone out in bioland used the Bio-Rad preparative electrophoresis
>cell (Model 491 Prep Cell)? The concept of continuous preparative
>electrophoresis sounds good, but I wonder if it is really practical on a
>*preparative* scale. Does anyone have an idea what the upper limit is on
>a single run in terms of amount of protein purified? 0.1 mg, 1mg, 10mg?
>>Thanks,
>>Mike Kennedy
>Dept. Biochemistry/Molecular Biology
>Mayo Foundation
>(kennedym at mayo.edu) "See this quarter? It used to be a nickel!"
Hi Mike,
Yes, I have actually used one of these things. My opinion is mixed.
The instrument worked, where all other methods failed, for the isolation and
purification of viral capsid proteins. I was able to get up to a couple of
mg/run.
It is theoretically possible to run very large quantities through the
device, but there is a fairly large amount of band spreading and as a
consequence you'll probably need to collect and pool fractions and re-run
them. Just as in a slab gel, you tend to get a bit of "smiling", the
difference is that it is radial in direction. In fact, the gel itself may
deform physically, due to the large amount of current run through it. There
is also a fairly large amount of dilution at the elution end.
It has been a few years since I used this thing, but if I recall
correctly, my set-up consisted of: a two head Gilson rabbit peristaltic pump
(two heads allow for regulating influx and elution of buffer so you don't dry
out and you dont flood your lab), a Gilson fraction collector, a Kipp and
Zonen chart recorder (with event marker input), and a Pharmacia UV detector.
I found that this relatively elaborate set-up WAS neccessary, and the cost of
these items needs to be factored into the total cost, fortunately we had a few
extra parts in the lab.
Pouring the gels takes a fair amount of practice, but you get the hang
of it. There is a fair amount of empirical monkeying around to get the gel
concentration right, things don't really work as well as a slab gel, and you
are trying to get the protein to elute out the other end, after all, so the
concentration of acrylamide is much lower than you would expect. Also, a half
percent or so change in concentration spells the difference between success
and failure (or at least a really LOOOOOOOOONG wait for elution).
I mentioned the fraction collector, the work really begins after the
run, because you have to analyze all the fractions (or at least a
representative number of them), to decide if you want them or not.
The folks at BioRad have done a pretty good job of putting together
protocols, so you may want to call them and ask them to send you some.
I developed a real love/hate relationship with this thing as I slaved
over it day after day, but we did eventually get enough protein to get on with
our work, so I guess it was worthwhile after all.
Good Luck.
George Trager
