resolving marginally different proteins on SDS-PAGE

Dr E. Buxbaum EB15 at le.ac.uk
Mon Sep 1 08:44:16 EST 1997


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.

To get better resolution, you want your protein get further down the gel,
so that small differences in Rf value get translated into bigger
differences in migration distance. Therefore you want a n acrylamide
concentration that allows the protein to migrate down as much as
possible. The use of longer gels (20 instead of 6 cm) can help. However,
if you use simple gels, longer migration distance means more diffusion
and therefore wider bands, which can offset the advantage gained. I had
this problem once and solved it by using a very flat acrylamide gradient
(5-7%) to counteract diffusion. Inclusion of 10% Sucrose in the heavier
solution stabilises the gradient during casting. Under these conditions
you can run your protein 3/4 down the gel without noticable band
spreading.




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