Hoefer TE22 - Manual (Page 14)

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sponge, and then place the membrane on the blot-
ting paper. Place the gel—which contains a sample
that has been electrophoretically separated and
equilibrated (if required) with transfer buffer—on the
membrane. Gently roll a glass pipet or test tube over
the gel to expel trapped air between the membrane
and gel. Cover the gel with a sheet of blotting paper
and then place a sponge of the proper thickness (see
the diagram below), again pressing gently to expel
trapped air.
4
Close the cassette and press lightly to lock the tabs.
The assembled cassette should hold the gel in firm
contact with the membrane without squeezing the gel.
If the stack seems loose, add sheets of blotting paper;
if the stack seems tight, replace the top sponge (over
the gel) with a sheet of blotting paper. If you remove
the bottom sponge (below the gel), substitute at least
two sheets of blotting paper to create space between
the membrane and the cassette panel.
one 3 mm sponge for gels
>1.5 mm
—OR—
one 6 mm sponge for gels
≤1.5 mm.
blotting paper
blotting paper
one 3 mm sponge
gel
membrane
Fig 2. Transfer stack assembly. The
stack is oriented so that negatively
charged molecules migrate toward
the grey anode (+).
Important! Do not overstuff the
cassette.
Note: Try to place the gel correctly
the first time because proteins
may begin to transfer immediately;
once transfer has begun, moving
the gel will distort results or cause
“shadow bands” on the blot.
The cassette panels are color
coded: black (top) = cathode side
grey (bottom) = anode side.
Assemble the cassette in a tray containing transfer
buffer about 3 cm deep.