Tim Magnuson (magn8831 at uidaho.edu) wrote:
: Hi Folks !
: I'm doing some work on an extracellular alkaline (pI>9) xylanase. Any
: reliable analytical native PAGE methods out there ? Also, any good
: western blotting buffer systems for alkaline proteins ?
A few years ago we were blotting a basic protein (also pI > 9)
to Immobilon P from SDS-slab gels and/or 2D gels. We had the same
question.
As I remember, we used a method included in the SemiDry BioRad
instruction manual, that's tris-glycine including SDS and methanol.
It is NOT the Towbin recipe, but rather a higher pH blend. It worked,
but I was always uncomfortable about less than complete removal of
protein from the slab gel: in other words, if you stained the slab gel
after blotting, there was a normal looking pattern, and that means a
lot of protein was left behind. But the blot looked fine too, whether
stained with Ab's or Coomassie blue.
It seems that people that use a tank blotter usually claim
100% transfer because the gel is devoid of protein. Actually they
only know 100% of protein is REMOVED from gel; it might be in the
buffer, etc.. Semi-dry users usually admit that proteiin remains in
the gel but that enough gets on the PVDF to do what you want. This
can be a problem if you are trying to be quantitative.
The method referred to is usually attributed to Khyse-Anderson
et al., or something close to that....
Good luck with it....
Steve Driska
