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.