IUBio

Electrophoresis problem

Kelly Luther umluther at cc.umanitoba.ca
Fri Mar 26 19:58:28 EST 1999


Andrew Warkman wrote:
> 
> > Do you mean stain the gel in coomassie, destain, and then electrophorese
> > the protein out of this same gel onto the nitrocellulose?  That wouldn't
> > work because the SDS would be leached during the staining and destaining
> > procedure, and the transfer of the proteins would be minimal at best.
> > The transfer would also be in both directions, and you would have to
> > place nitrocellulose on both sides of the gel when you did the
> > electrophoresis.
> 
> Not true..
> 
> (1) if SDS is in the sample loading buffer (and assuming that the samples
> are boiled before loading) the SDS will be permanently bound to the proteins

Do you have a reference for this.  I am intrigued as my statements were
based on my own experiences and understanding of the mechanisms involved
in these protocols.

> 
> (2) SDS bound to the protein serve to masks the native charge and gives the
> entire protein a negative charge (with the same chare to mass ratio) thus
> all protein would be electrophoresed (I prefer the term  electroblotted)
> toward the positive electrode
> 
If the SDS remains on the protein you are correct.

I still don't agree that the SDS isn't leached away.  I tried to leave a
gel in transfer buffer overnight prior to electrophoreses onto
nitrocellulose, no protein transfered.  I had anticipated leaching of
SDS and had increased the percentage of SDS in the transfer buffer that
I had put the gel into.  The proteins did not diffuse from the gel in
any noticeable amount when compared to a twin gel after electroblotting
so I concluded the SDS must have leached away.

There is also one more problem.  When staining with coomasie blue you
first have to fix the proteins into the gel.  The staining solution is
designed to do just that.  I can't see how they are then electrophoresed
from the gel once fixed.

Have you managed to blot a coomassie stained gel?  I would be interested
in all of the details if you have.

Regards

-- 
******************************************************************
Kelvin Luther
Department of Chemistry         "Illegitimati non carborundum"
University of Manitoba            General Joseph W. Stilwell
Winnipeg, MB, Canada
mailto:umluther at cc.umanitoba.ca
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