Hoefer SE640 - Manual (Page 19)

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p11
Acrylamide gels
1
Prepare the monomer solution and pour the gel
See Appendix A for SDS-PAGE recipes. Prepare the
required amount of monomer solution. De-aerate and
add the initiator (ammonium persulfate, APS) and
catalyst (TEMED) just prior to pouring the gel. Pipet
the solution into one corner of the sandwich, taking
care not to introduce any air bubbles. See below
for the appropriate solution level according to the
application.
No stacking gel (Continuous system)
Fill solution to just below the top of the upper plate
edge. If bubbles are trapped, remove with a pipette or
syringe. Introduce a comb (at a slight angle) into each
sandwich, taking care not to trap air bubbles under
the teeth.
Club sandwich
Pipette 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
2). After the gel is set, prepare the stacking gel as
described below.
2-D electrophoresis (Discontinuous protein system)
Fill monomer solution to about 1 cm below the top
of the glass plate to allow 4 – 5 mm for the IPG strip
or tube gel and an agarose seal. (A stacking gel will
require extra space). Seal the IPG strip or tube gel in
place with agarose dissolved in running buffer. Take
care to avoid trapping any air bubbles between the
first- and second-dimension gels.