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Acrylamide Polymerization A Practical Approach

temperature (for systems in which constant agitation is used during degassing, 10 min is sufficient). Longer periods of degassing are generally not deleterious, although long degassing will result in somewhat faster polymerization.


Chemical Polymerization in Discontinuous Systems Lower (Resolving) Gel
In a discontinuous system, such as that of Laemmli, the resolving gel is polymerized first. Then, the stacking gel is cast on top of the resolving gel. Use the following protocol to prepare resolving gels for all discontinuous systems (see the next section for preparation of stacking gels).

Swirl the solution gently but thoroughly. Holding the flask by the neck with one hand, swirl it 8 to 10 cycles. This mixes the initiators completely without introducing too much oxygen. Swirling too little can result in uneven polymerization.

Cast the gel by introducing the monomer solution into the gel mold in a steady stream to minimize the introduction of oxygen. Overlay the monomer solution using water, isoamyl alcohol, or water-saturated isobutyl alcohol to exclude oxygen from the surface.

Allow polymerization to occur at room temperature at least 90 min prior to use (see Figure 1).

Chemical Polymerization in Continuous Systems and Stacking Gels
Continuous systems consist of a single gel. Continuous systems are used for some types of protein electrophoresis, and for DNA sequencing. Stacking gels are part of discontinuous systems. These gels have in common contact with the well-forming comb and greater exposure to molecular oxygen at the surface. Use the following levels of initiators for continuous systems and stacking gels.
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