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., Electro phoresis 1992, 1,3, 587-595) is not inhibited by high ionic strength n or in the pH 4-10 interval. However, the bundles are disaggregated by glycerol (T(m) at 20%) and by ethylene glycol (T(m) at 24.5%) as well as by pH extremes (pH 3 and pH 11). Supercoiling is also strongly inhi bited in a copolymer, formed by acrylamide an N,N-dimethylacrylamide o r 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 th e above data strongly suggest that the nascent chains are held togethe r in bundles by hydrogen bonds prior to the cross-linking event, inste ad of having a random orientation and distribution in the solvent. How ever, it is not possible to distinguish between H-bonds oriented perpe ndicular to the chain axis vs. H-bonds occurring within a single polym er filament, and the two types of H-bonds probably coexist. Macroporou s gels perform well in steady-state electrophoretic techniques, such a s conventional isoelectric focusing and immobilized pH gradients, wher e a large-pore structure is necessary for fast protein migration and f or attainment of equilibrium conditions.