another GST story #2

Dietmar.Tietz at agrar.uni-giessen.de Dietmar.Tietz at agrar.uni-giessen.de
Wed Jul 20 01:08:50 EST 1994


> >
> >I have been under the impression since my grad school days that proteins
> >(after boiling in the presence of 2-mercaptoethanol and SDS) attain a
> >relatively unfolded configuration and run as rods on SDS-PAGE.  I thought
> >that this was the reason that sizes of proteins can be compared using the
> >relative mobility on SDS-Page gels as opposed to native gels:  All the
> >proteins have essentially the same shape as well as charge to mass ratio
> >(due to the SDS).  I'd appreciate knowing if my impressions are incorrect.
> >
> >***************************************************************
> >Stephen R. Lasky, Ph.D.       Brown University/Roger Williams Medical Center
> >e-mail: Stephen_Lasky at brown.edu         LandLine: 401-456-6572
> >~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
> 
In general it is true that SDS treatment destroys the quaternary and most
of the tertiary structure.  However, the denatured proteins assume something
like a random coil, not a rod-like structure, thus having a more or less
spherical shape.  But also many of the native proteins, except for fibrinogen,
etc., are pretty spherical.  The benefit for electrophoresis, however, really
is that it helps dissolve some otherwise really insoluble proteins and, most
of all, provides them with a uniform surface net charge density.  Since gel
electrophoresis depends on both particle charge and size, one would be 
otherwise not able to determine sizes from just a SINGLE gel.  If size and
charge varies, it can be compared with solving ONE equation with TWO
unknowns.

The unfortunate thing is that SDS does not bind at the same ratio to all
proteins and there is no way of detecting it on ONE gel.  That is the
reason that size determinations are sometimes off from the correct value.
If you want to be really sure, do Ferguson plots.  I have explained that
a few days ago on the network. 


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*           Dietmar Tietz, Ph.D.,  Research Scientist            *
*            Biostatistics, Justus-Liebig-University             *
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*                  Phone: +49-(641)-702-6015                     *
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