4
3.3
Removing the Gel
1. Turn the power supply off and disconnect the leads
from the power supply. Remove the safety cover
from the unit, by simultaneously pressing down on
the white push pins with your thumbs, while lifting
up on the yellow safety cover with your fingers. See
Figure 2.
Do not remove safety cover by pulling
up on leads!
2. Pull up on gel door latches, and open gel door.
Remove gel cassette.
3. Fix and stain gel according to your preferred
method.
Section 4
INSTRUCTIONS FOR WESTERN BLOTTING
4.1
Preparing the Unit for Blotting
1. Remove safety cover from the assembled unit by
simultaneously pressing down on white push pins
with thumbs while lifting up on yellow safety cover
with your fingers. See figure 2.
Do not remove
safety cover by pulling up on leads!
Remove
white core from lower reservoir by grasping core with
one hand and lifting directly up. Open doors on the
core assembly by pulling up on the white latches.
2. Open blotting cassette (as shown in figure 8) and lay
it flat on the bench.
3.
Western Blotting Instructions.
a.
Soak gel in 1X Towbin (1X tris-glycine buffer/
20% Methanol) buffer for 10 minutes.
b.
Cut a piece of PVDF or nitrocellulose membrane
to fit the gel.
Do Not Touch Membrane with
Bare Hands. Wear gloves.
c.
Soak membrane in 50% methanol for 30
seconds.
d.
Soak membrane in water for 5 minutes.
e.
Soak membrane 5 minutes in 1X Towbin buffer
until it no longer floats.
Do Not Allow
Membrane to Dry.
f.
Cut 1 piece of filter paper 1 mm thick or less to fit
the gel.
g.
Soak sponge in 1X Towbin Buffer.
4. Assemble blotting stack. With blotting cassette wide
open assemble components on black side in the
following order, as shown in Figure 9: foam pad,
gel*, buffer saturated transfer membrane, then buffer
saturated blotting paper. Smooth with gloved finger
or roll with glass rod to be sure no bubbles exist
between the gel and the transfer membrane.
Figure 9
*NOTE: to prepare gel for blotting, trim off wells and
any excess gel at the bottom, and invert 180º so that
the large molecular weight proteins are at the
bottom of the cassette. This puts the large proteins
in contact with a stronger field strength and allows
the blotting transfer to take place more efficiently.
5. Insert blotting cassettes into core making sure that
red side faces outward, as shown in figure 10.
Figure 8
Red Side
Figure 10
