Efficiency of electroelution?

Dr Engelbert Buxbaum engelbert_buxbaum at web.de
Tue Nov 5 05:57:00 EST 2002


dustjohn at hotmail.com wrote:

> Just wondering if electroeluting proteins (i.e. cutting out protein band
> from native PAGE and passing current through the gel slice) is a decent
> way of isolating pure protein?  Does this method sacrifice enzymatic
> activity for purity or can the enzymatic activity be saved?  (I hear one
> must avoid production of free radicals within the buffer?)

After SDS-PAGE the protein will most likely be dead, as you are working
under denaturing conditions.

With native PAGE (Ornstein) or native blue PAGE (v. Jagow) you might get
lucky. 

There are other detergents like CTAB, which can solubilise proteins in
an active state. You may with to try, just remember to change the
polarity of your power source as proteins now move to the negative pole.

Regarding free radicals storage of the gels o/n in the fridge before
using them probably takes care of most of the problem.

You do not need to do electroelution. It is possible to cover the bottom
of the gel with dialysis membrane and slowly pass running buffer between
the membrane and the gel. The proteins will then migrate into this
buffer one after the other. BioRad offers a preparative electrophoresis
unit, never used it though.



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