Experience with BioRad Rotofor system
jproudman at ASRR.ARSUSDA.GOV
Thu Mar 3 22:21:58 EST 1994
-John <newittja at delphi.com> writes:
>"Steve A. Kay" <sak5y at FARADAY.CLAS.VIRGINIA.EDU> writes:
>> Has anyone had any experience with the BioRad Rotofor
>>and/or Prep Cell protein purification systems ? We are
>>considering purchasing one and would appreciate any comments.
>I tested the Rotofor once with an E. coli lysate from cells ex-
>pressing my protein at moderate levels. A MAJOR problem with
>this apparatus is that once the proteins near their pI, they
>precipitate. I tried several of the suggested workarounds, but
>I never had any success. We didn't buy the apparatus.
And Andrew <wallace at irbm.it> wrote:
>A student in our lab tried the apparatus and had exactly the same
>problem. We didn't buy it either.
I beg to differ with these opinions. The results pointed out by the above
experiments points out the ADVANTAGE of the Rotofor! MANY proteins
precipitate when they near their isoelectric point- this is not the fault
of the apparatus. One limitation of column IEF is that such precipitates
then fall through the pH gradient and mess up the purification. The design
of the Rotofor (rotating horizontal column with baffels to prevent mixing)
keeps precipitates isolated. You can then elute the fraction containing
your protein, redissolve, and refocus in a narrower gradient or use in
another technique. Of course, if precipitation of your protein denatures
it, then you should probably not use ANY IEF apparatus.
As for my experience with the Rotofor, it worked well in purifying my
protein DESPITE precipitation of other contaminating proteins present
in the crude sample.
No connection to Bio-Rad- just my experience.
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