A new buffering system and its use in electrophoresis and isoelectric focusing.

A novel buffer system with rather unique properties particularly well suited for preparative isoelectric focusing is described. The system was developed in response to the long standing need for pH gradients formulated using inexpensive components of known chemical composition. The system is binary consisting of buffer pairs covering the pH range of 3 to 10, in increments of less than 1 pH. The components are all readily available, nontoxic, and biologically acceptable, and are selected on the basis of their dissociation constants. The requirement for each buffer pair is that there be an overlapping pH zone where both components are largely non-ionized, having a ratio of neutral species to total buffer concentration between about 0.90 and 0.99. This ratio is defined as the 'electrophoretic reserve capacity' (ERC). Within the above ERC range a stable pH gradient is obtained, assuring sharply resolved protein focusing. Buffer parameters, mainly pH, buffering capacity, ERC, transport numbers, and conductivity, can be easily calculated and the buffer composition adjusted to meet requirements. All components are used at high concentration, typically of the order of up to 100 mM, yielding buffering capacities and conductivities analog to those customary in electrophoresis.