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2.2. Acrylamide gel preparation
Table 1. Approximate monomer solution volume
required for a single gel
Gel thickness (mm)
Model
0.75
1.00
1.5
SE 400
15 ml
23 ml
30 ml
SE 410
23 ml
34 ml
45 ml
2.2.1. resolving gel
1��
Prepare the monomer solution and pour the gel.
Prepare the required amount of monomer solution,
deaerate, and add the initiator and catalyst just prior
to pouring the gel.
2��
Pipet the solution into one corner of the sandwich,
taking care not to introduce any air bubbles. See
below for the appropriate solution level:
No stacking gel
(Continuous system.) Fill solution to just below the
top of the upper plate edge. If bubbles are trapped,
remove with a pipet or syringe. Introduce a comb (at
a slight angle) into each sandwich, taking care not to
trap air bubbles under the teeth.
2-gel sandwich
Pipet the solution into both sandwiches, filling each
to the same level below the notched edge.
Stacking gel
Fill solution to 3–4 cm below the top of the glass
plate. This height allows 1 cm of stacking gel below
the wells. Pour the gel and apply an overlay (see step
3). After the gel is set, prepare the stacking gel as
described in the next section.
2-D electrophoresis
(Discontinuous system) For the second dimension
resolving gel, fill solution to ~1.0 cm below the top of
the glass plate (leave extra space for a stacking gel, if
required). One centimeter allows enough space for the
first dimension IPG strip or tube gel and an agarose
seal. (While transferring, take care to avoid trapping
air between the tube gel and slab gel; seal the tube
gel into place with agarose in electrophoresis buffer.)
Note:
Appendix A lists recipes
for the Laemmli gel system.