Electrophoresis problem

Kelly Luther umluther at cc.umanitoba.ca
Tue Mar 23 23:53:03 EST 1999


Holger Neumann wrote:

> 
> I personally think you have problems transferring your protein to the
> nitrocellulose membrane.
> Perhaps this can help:
> 1. run SDS-Page
> 2. stain SDS with Coomassie
> 3. document your stain on photo (digital or conventional)
> 4. destain SDS-Page
> 5. Western blot proteins
> 6. stain membrane with Ponceau
> 7. document this second stain like the first one (see 3.)
> 8. evaluate both pictures to see where the problem occured
> 
> if bands are smearly in Coomassie stain your SDS run makes some problems
> if bands are clear in Coomassie but smearly in Ponceau stain you have problems
> transferring the protein
> 
> Good luck

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.

-- 
******************************************************************
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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