Alternatives to Laemli?
nobody at elnino.com
Mon Feb 16 17:55:05 EST 2004
"MICHAEL L SULLIVAN" <mlsulliv at facstaff.wisc.edu> wrote in message
news:4b33fd4b0021.4b00214b33fd at wiscmail.wisc.edu...
> > lab
> > gels. Since there is
> > > no stack with this one, sample volumes need to be small to get
> > tight bands.
> > There is no reason not to include the stacker.
> Is there a point to including a stacking gel here, since the pH difference
wouldn't be that great between the stacking/resolving gels? I thought that
was the point of a stacking gel-- having the pH difference at the interface
of the resolving/stacking gel. Of course, I've been puzzled how one can
have precast gels, since presumably the pH difference couldn't be maintained
upon storage, so maybe I'm wrong on this.
The Laemmli stacking system works by utilizing a difference in mobility of
glycine and SDS/protein at different pHs. At pH 6.8 glycine and proteins
have relatively equal mobilities through low percentage PAAG. However, when
both reach separating gel at pH 8.8, glycine picks up creating a charge
"vacuum" zone immediately behind the transition line between the two gel
layers. This charge vacuum slows down migration of proteins as they enter
the separating gel, causing a stacking effect. I hope I remember that
As for the original post, I don't think SDS-based buffer would work for
studying protein/RNA complexes at all, simply because SDS would denature the
protein. There are non-denaturing buffer systems, like Tris-borate-EDTA
(TBE) that work fine for that purpose.
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