resolving marginally different proteins on SDS-PAGE

Howard Petrie h-petrie at ski.mskcc.org
Fri Aug 29 16:52:20 EST 1997


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.

Howard Petrie
Assistant Member, Immunology Program
Memorial Sloan-Kettering Cancer Center
h-petrie at ski.mskcc.org



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