Acidic polyacrylamide gels

Robert Horton horton at molbio.cbs.umn.edu
Mon Sep 21 12:24:16 EST 1992


I received the following response to my earlier post. I thought I'd pass it on
to the Net so that my fellow molecular biologists might become enlightened as I
have been.

(me)
>>Pardon my ignorance (I'm one of those "molecular biologists" who practices
>>biochemistry without a license), but couldn't you just hook the electrodes up
>>the other way? A basic protein should migrate towards the negative electrode
>>...?

(CURT ASHENDEL <ashendel at bchm1.aclcb.purdue.edu>)
>        Are you kidding???!!  They should take away your license.
>Discontinuous gels require a very specifc pH dependent stacking to get their
>high resolution.  This depends on the stacker gel ion transfer (using chloride)
>to be faster than the glycine in the electrode buffer.  Running it with
>inverted polarity will cause the chloride to go up, along with the SDS, and the
>whole thing would not stack or run at all.  Now if it were a non-discontinuous
>gel without SDS, sure it would work.  Actually, highly basic proteins run fine
>in SDS gels (witness histones and meylin basic protein and protamine), it is
>just that they bind an unusually low amount of SDS for their size and tend to
>migrate at the wrong apparent molecular weight.  If a basic protein is not
>running right, try increasing the SDS conc.
>
>        I know you didn't ask the question, but I couldn't resist the chance to
>spread a little biochemistry.  I guess it is easier to learn molecular biology
>than it is to learn biochemistry.
>
>Curt Ashendel
>Purdue Univerity

   First, I already said I don't _have_ a license.
   Second, I thought the operative adjective was "native" (=> no SDS).
   Third, nobody actually said anything about a discontinuous gel. Apparantly,
not everyone uses them, e.g. bertheau at inapg.inra.fr, who wrote:
>Several years ago I used for alkaline proteins a Triethylamine HCl buffer:
>pH 11, it worked well in native conditions...
> -Yves Bertheau
Since this buffer uses a chloride anion, it seems like it would not work for
stacking?
   Finally, [vicious flame about reading for comprehension being more difficult
to learn even than biochemistry omitted.]
--
Bob Horton            /\ "Crash programs fail because of the theory that
U. of Minnesota, CBS  || with nine women pregnant you get a baby a month" 
1479 Gortner Ave.    /||\   -Werner von Braun.  Disclaimer:"Bob who?"
St. Paul, MN 55108    ^^   horton at molbio.cbs.umn.edu/(612) 624-3790



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