Western Blot

Pamela Norton pnorton at lac.jci.tju.edu
Wed Aug 30 17:26:57 EST 1995


In article <41v146$9dt at fiona.umsmed.edu>, hutchins at fiona.umsmed.edu (Jim
Hutchins) wrote:

> Alison Weaver (a.weaver at auckland.ac.nz) wrote:
> : I am having problems with the transfer of a 150KDa protein which 
> : has a transmembrane domain from SDS PAGE to Hybond C membrane. 
> : Does anyone have any suggestions how transfer may be enhanced? 
> 
> Along with the suggestions of others (lower MeOH, maybe some SDS) you
> might also try two other things:
> 
> 1. Blot in 10 mM CAPS buffer (Matsudaira, 1989)
> 
> 2. Use a different membrane, e.g. PVDF.


    My curiosity has been sparked by this thread. I don't have extensive
experience with blotting many different types of proteins, but we routinely
transfer proteins of >200 kDa in 20% MeOH, no SDS. How much is transfer
affected by the hydrophobicity of the protein in question? (ours are not
very). Is there a good reference for this sort of thing?

				We also use low percentage gels (6-7%, gooey). I have noticed poor
transfer of large proteins out of higher percentage gels. Alison, what %
gel are you using?



-- 
Pamela A. Norton, Ph.D.          Assistant Professor of Medicine
Thomas Jefferson University
Philadelphia, PA 19107           p_norton at lac.jci.tju.edu



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