Bio-Rad Preparative Electrophoresis Cell

xyzzyx xyzzyx at aimnet.com
Fri Jul 7 00:07:20 EST 1995


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





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