Blue-Native Gel Electrophoresis and Western Blotting

wulf-dirk.leuschner at tuebingen.mpg.de wulf-dirk.leuschner at tuebingen.mpg.de
Sun Nov 30 12:29:54 EST 1997


Hi there,

does anyone out there has experience in blotting of blue native gels?

We've got the following problems:

(1) Migration of marker proteins: Since different proteins bind
different amounts of the Coomassie dye the question arises whether
this has dramatic consequences on the migration behaviour. E.g.
ferritin (440 kDa) does not seem to bind Coomassie and seems to
migrate more slowly compared to other - dye binding - proteins of the
same Mr. However, people use ferritin as a molecular weight marker in
BN-PAGE...

(2) 'Ghost' bands in marker lane: Using different marker proteins we
sometimes find quite a big 'blob' of a signal in our western blot no
matter what sort of protein we take as a marker. It appears to have a
Mr between 150 and 440 kDa...

(3) Blotting efficiency: Coomassie seems to inhibit protein binding to
PVDF (or NC) membranes. This might be due to the fact that the dye
migrates rapidly through the gel and then binds to the membrane before
even the first protein molecules arrive there. Does anyone know how to
get rid of Coomassie in the gel (before blotting) without
precipitating the proteins in the gel?

Hope there are a few people who already solved these problems...

WDL.




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