Acrylamide Polymerization A Practical Approach ( nd changes in buffer pH. Exce...)

Acrylamide Polymerization A Practical Approach

nd changes in buffer pH. Excess TEMED can increase buffer pH, react with proteins (Dirksen and Chrambach 1972; Chrambach et al. 1976), and alter the banding pattern (Gelfi and Righetti 1981a). Ammonium persulfate acts as a buffer between pH 8 and 9. Potassium persulfate is recommended instead of ammonium persulfate in weakly buffered basic systems (~pH 9). Excess riboflavin may cause the oxidation of some compounds, especially sulfhydryl-containing compounds (Dirksen and Chrambach 1972), and can denature proteins (Righetti et al. 1981).

Reducing the concentration of initiators results in longer polymer chain lengths, lower turbidity, and greater elasticity. These are desirable properties. However, lower initiator concentrations also mean slower polymerization. If polymerization is too slow, oxygen will begin to enter the monomer solution and inhibit polymerization, resulting in gels which are too porous and mechanically weak. Inhibition will be especially pronounced at surfaces exposed to air, or at the surfaces of combs and spacers, which appear to trap air at their surfaces. The remaining unpolymerized monomer can react with alpha amino, sulfhydryl, and phenolic hydroxyl groups of proteins (Allison et al. 1974; Chrambach et al. 1976; Dirksen and Chrambach 1972).

For discontinuous systems which employ a stacking gel (e.g., Laemmli system), optimal polymerization of the overlaid lower gel (resolving gel) is achieved when visible gelation takes place 1520 min after the addition of the initiators ammonium persulfate and TEMED (note that polymerization continues long after visible gelation takes place; see Figure 1). For stacking gels and continuous systems (which do not
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