Hoefer TE42 - Manual (Page 17)

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p9
Transfer
Take care in orienting all system components so
that the electric field applied causes all species
to migrate toward the membrane. The migration
direction depends on both the characteristics of
the sample and the pH of the transfer buffer. If
the species of interest is negatively charged
in the transfer buffer and the stack is assembled
so that the membrane is nearest the grey side
of the cassette, then this side faces the anode
(+). Most proteins migrate toward the anode in
the Towbin Tris/glycine/methanol buffer system
(independent of the presence of SDS), and under
most conditions nucleic acids are negatively
charged and also migrate toward the anode.
Cooling is strongly recommended. Any setting
that results in higher than 5 W of power will
generate enough heat to require active heat
control. A refrigerated circulator bath should
be set to cool to about 10 °C. (If using 50/50
ethylene glycol/water, the temperature can be set
lower.) Chill buffer before use if possible.
Recommended power settings. Most transfers
are complete within one hour, but larger mole-
cules or thicker gels may require longer transfer
times; the optimum transfer time for each system
must be determined empirically. Transfers left to
run overnight should be set to a constant current
setting no higher than 0.1 A.
Important! Never allow the buffer
temperature to exceed 45 °C.
Excessive heat will cause the unit
to warp.
Typical transfer parameters
Parameters for your sample and buffer system
must be determined empirically.
protein nucleic acids
Buffer Towbin 1X TBE or 1X TAE
Current (A) 0.8–1.0 0.9–1.0
Voltage (V) 70–80 50
Transfer time ~1 hour ~1 hour
Coolant temp. 10 °C 10 °C or less