resolving marginally different proteins on SDS-PAGE

Ian McFarlane I.McFarlane at icrf.icnet.uk
Wed Sep 3 05:23:58 EST 1997


In article <newitt-ya02408000R0209971452090001 at news.nih.gov>,
newitt at removeme.nih.gov (John A. Newitt) wrote:

> In article <h-petrie-2908971748590001 at news.ski.mskcc.org>,
> h-petrie at ski.mskcc.org (Howard Petrie) wrote:
> 
> > We occasionally need to resolve hyper- and under-phosphorylated forms of a
> > protein (pRb) on SDS-PAGE gels for Western blot analysis.  Individuals
> > familiar with these isoforms will know that they differ by an apparent
> > (maximum of) 6 Kd, and separation is tricky.  I'm wondering what those of
> > you with an interest in the theoretical aspects of SDS-PAGE think about
> > the use of higher versus lower relative percentages of acrylamide for such
> > applications.  I know that lower percent gels (e.g., 5-7%) are routinely
> > prescribed for efficient resolution of proteins in this size 'range.' 
> > However, I'm not really interested in resolving pRb isoforms from other
> > proteins, since Western blotting is (relatively) specific.  For the
> > efficient resolution of proteins with marginally different migration
> > coefficients, wouldn't a higher (e..g., 10%) percentage of acrylamide work
> > better?  Your thoughts are appreciated.
> 
> I've always found that I get the best resolution of similarly migrating
> species using a straight percentage gel and adjusting the acrylamide
> concentration such that the desired bands migrate to the center of the
> resolving gel (i.e. Rf=0.5).
> 
> Regards,
> 
> John A. Newitt, Ph.D.           |   <newitt at removeme.nih.gov>
> National Institutes of Health   |   FAX: 301-402-0387
> Bethesda, Maryland  USA         |   
> 
> Please delete "removeme." from my address to reply by email.

I've been told that running gels in 6M urea (deionised) will sharpen the
bands considerably. However I don't have any refs on this.

Ian Mc



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