liquid or semidry western blotting?

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Tue Oct 3 08:52:52 EST 2000


I'm in a similar situation, that is, we have a large format transfer
apparatus, which does seem sort of silly for transferring 1 minigel!

That said, I prefer wet to semidry transfer.  In my last lab, several
people did side by side comparisons, and liquid transfer always seemed to
give better and more consistant results.

Now, as for the huge buffer tank, I've been using an old Hoeffer set up,
which I think in theory should hold 5 l of buffer.  I've been making 4
liters (1X tris-glycine, 20 % MeOH) and reusing it 4 times.  Reusing it
doesn't seem to be a problem.  I just put it in a jug and put it in the
cold room.

I've been transferring for 2 h. at 500 mA current with the unit on ice and
a stir bar in the buffer.  I've not optimized transfer or anything and I
know there's still a fair amount of protein in the gel.  However, when I
stain, I can see that there's also a lot of protein on the membrane.  The
whole set up is a bit of a hassle, but we're not really doing enough
blotting right now to justify a new minigel unit.

Hope this helps,

Mike

Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264)-5147 Fax


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