2D Electrophoresis sample prep

Maca maca_30 at hotmail.com
Mon Dec 7 22:28:32 EST 1998


Andrew and Glen,

The presence of a small amount of SDS in your sample preparations is fine and will
not result in perturbation of your proteins' apparent pIs from your gel.  In fact,
as Andrew has found, including it can improve your final results.  Boiling of
samples in SDS is sometimes the only way to stop proteolysis in your sample.
Proteolysis is highly undesirable in 2D gels as it leads to artefacts.

The trick is to keep the SDS at a concentration that is 4 times lower than the
concentration of the non-ionic detergent (I assume that you're using CHAPS).  At
these types of concentrations, CHAPS will sequester the SDS; forming micelles and
taking the charge from the solubilised protein.  You can work out these things
empirically if you look at the Critical Micellular Concentrations (CMCs) of the
detergents that you're working with and the temperature that you do the
experiments.

Some references for you: (The Rabilloud paper is a "bible" for 2D gels.  Look at
the first paragraph on p825 for the SDS problem).

Rabilloud, T.  Electrophoresis 17 1996 Solubilization of proteins for
electrophoretic analyses pp 813-829.

Ames G.F.L., Nikaido K. Biochemistry 15 1976 pp616-623.

Weber, K, Kuter D.J., J. Biol. Chem 246 1971 pp4504-4509.

Remy R, Ambard-Bretteville, F., Methods Enzymol 148 1987 pp623-632.

Hope this helps - post back to the group with your thoughts!
Maca.


Glen Tamura wrote:

> Andrew:
>
> I agree with you. There is very little question in my mind that this will
> result in alterations in the apparent pI. On the other hand, if the amount
> of SDS which binds is constant, you might still get nice separation. I
> certainly would use any pI information from such gels with a very large
> grain of salt (or is it a small grain?).
>
> Glen Tamura
>
> On Thu, 26 Nov 1998, Andrew Pridmore wrote:
>
> > I have achieved excellent two-dimensional separations of bacterial proteins
> > using a sample prep described in a Japanese paper, which includes SDS as
> > well as nonionic detergent in the first dimension sample buffer.
> >
> > Why does this work?
> >
> > Surely the negative charge imparted to the proteins by the SDS would disrupt
> > the isoelectric focussing? Or does the nonionic detergent displace the
> > negative charge?
> >
> > Does anyone have a reference which demonstrates what is happening?
> >
> > Thanks for reading this!
> >
> > Andrew Pridmore
> >
> >
> >
> >




More information about the Proteins mailing list