resolving marginally different proteins on SDS-PAGE

John A. Newitt newitt at removeme.nih.gov
Tue Sep 2 14:52:09 EST 1997


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.



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