( active in initiation) actual...)Acrylamide Polymerization A Practical Approach

This explains why polymerization initiated primarily by riboflavin can be completely blocked by exhaustive degassing. However, oxygen in excess of that needed to convert riboflavin to the active form will inhibit polymer chain elongation, as it does in reactions initiated only by ammonium persulfate and TEMED. Thus, while degassing is still important for limiting inhibition, it must not be so extensive that it prevents conversion of riboflavin to the active form. For polymerization initiated by riboflavin/TEMED, or riboflavin/TEMED/ammonium persulfate systems, degassing should not exceed 5 min.

A consequence of the interaction of riboflavin with oxygen is that riboflavin seems to act as an oxygen scavenger. This is supported by the observation that the addition of riboflavin (5 g/ml) to stacking gel solutions containing ammonium persulfate/TEMED initiators results in cleaner, more uniform polymerization at gel surfaces exposed to oxygen (such as combs). The same effect could likely be achieved by more thorough degassing of solutions without riboflavin.

Whether using chemical polymerization (ammonium persulfate/TEMED) or photochemical polymerization (riboflavin/TEMED or riboflavin/TEMED/ammonium persulfate initiators), reproducible gel quality and separation characteristics require careful attention to gel solution temperature before degassing, and to degassing time, temperature, and vacuum. These parameters should be kept constant every time gels are prepared.

pH
The majority of electrophoresis systems are buffered at neutral or basic pH, at which the common initiators, ammonium persu
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