Polyacrylamide gel electrophoresis (PAGE) provides a versatile, gentle, high resolution method for fractionation and physical-chemical characterization of molecules on the basis of size, conformation, and net charge. The polymerization reaction can be rigorously controlled to provide uniform gels of reproducible, measurable pore size over a wide range. This makes it possible to obtain reproducible relative mobility ( R f ) values as physical-chemical constants. Application and extension of Ogston's (random fiber) model for a gel allows for calculation of molecular volume, surface area, or radius, free mobility, and valence from R f measurements at several gel concentrations, to calculate gel concentration for optimal resolution, and to predict behavior of macromolecules on gel gradients by computerized methods. Extension of classical moving boundary theory has been used to generate multiphasic buffer systems (providing selective stacking, unstacking, restacking, and preparative steady-state-stacking) with known operating characteristics for any p H at 0° and 25°C. A general strategy for isolation of macromolecules and for macromolecular mapping has been developed. Preparative scale PAGE is operational for milligram loads and feasible for gram quantities.