In Article <4hhheo$afa at lyra.csx.cam.ac.uk>, cdw1000 at mole.bio.cam.ac.uk
(Christopher Walentas (Bioc)) wrote:
>I was wondering if anyone had a buffering system that they could
>recommend for the resolution of two proteins using native PAGE at
>a pH at or below 6.5? I've got a mixture of two species which
>differ by a series of histidines, and need to utilise their charge
>differences (which are only relevant below their pKa), but cannot
>go too acidic due their pI is around 4. I've tried citric acid
>at pH 5.5 (100mM) but with not much luck-- the proteins are 100kDa
>and hardly made it into the resolving gel by the time the BPBlue
>had scooted off the bottom.
Two points tha might be helpful.
1) If possible, lower your acrylamide concentration (but no lower than 5%)
to increase the protein's mobility in the gel.
2) There is nothing magic about BPBlue going off the end of the gel. Just
run longer (use a timer). You already know how far your bands migrate into
the gel in the time it takes for the BPBlue to run off, just multiply that
time by gel length divided by the migration distance of the fastest
These two points will help you maximize the separation using the citric acid
Department of Biological Sciences, University of Alberta
wgallin at gpu.srv.ualberta.ca