Agarose gels for protein purification?
houthaeve at embl-heidelberg.de
Tue May 10 12:18:54 EST 1994
In article <berk-050594154934 at koniskyj3.life.uiuc.edu>, berk at uxa.cso.uiuc.edu (Holger Berk) writes:
> My question THIS time is about using agarose gel electrophoresis for the
> purification of proteins. Has anyone out there had any experience with
> this procedure? Is it possible to detect the proteins in the agarose gel
> by coomassie blue staining, or do you have to transfer the protiens to a
> membrane first? Also, is contamination with proteins present in the
> agarose itself a problem? Any references anyone could send me about this
> procedure would be very greatly appreciated!
> Thank you,
> Ed Beaty
> Ed_Beaty at qms1.life.uiuc.edu
You hit a hot point.
Let's put it like this : running in agarose gives problems as agarose is
not keeping the pH as good as acrylamide does.
However by pooring first a acrylamide supporting gel and then a short
agarose separating gel, followed again by a separating + stacking gel,
you do obtain the best results.
Coomassie staining is no problem. Transfer/blotting should also not be
difficult (cfr. Southern/Northern DNA-blotting).
The purity of the agarose is still a problem.
If you 'd like to for further analysis on the mass spectrometre, you might
have to face problems with impurities.
If it's just for The Edman sequencer or the amino acid analyser, these
machines can live with the problems. [performing of course intensive washing
on both machines ].
However a short RP-HPLC step in before can solve problems...
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