Conventional isoelectric focusing and immobilized pH gradients in 'macroporous' polyacrylamide gels

Lateral aggregation in presence of a hydrophilic polymer (e.g. 10 kDa polyethylene glycol) in the gelling solution (Righetti et al., Electrophoresis 1992, 13, 587-595) is not inhibited by high ionic strength nor in the pH 4-10 interval. However, the bundles are disaggregated by glycerol (Tm at 20%) and by ethylene glycol (Tm at 24.5%) as well as by pH extremes (pH 3 and pH 11). Supercoiling is also strongly inhibited in a copolymer, formed by acrylamide an N,N-dimethylacrylamide or N-methylacrylamide. A level of 50% uncoiling is obtained well before a 1:1 ratio, already at a level of 18% N,N-dimethylacrylamide. All the above data strongly suggest that the nascent chains are held together in bundles by hydrogen bonds prior to the cross-linking event, instead of having a random orientation and distribution in the solvent. However, it is not possible to distinguish between H-bonds oriented perpendicular to the chain axis vs. H-bonds occurring within a single polymer filament, and the two types of H-bonds probably coexist. Macroporous gels perform well in steady-state electrophoretic techniques, such as conventional isoelectric focusing and immobilized pH gradients, where a large-pore structure is necessary for fast protein migration and for attainment of equilibrium conditions.