Pore gradient electrophoresis

Abstract

Gel gradient electrophoresis has been proposed as a method for improving resolution and for measuring molecular size. Margolis and Slater have suggested that each macro-ion will reach a “pore limit” or “dead stop” and that the protein pattern would remain essentially stable thereafter. However, no quantitative treatment of the migration of charged macromolecules in a gel gradient has been reported to date. The present report provides an analysis of the behavior of macro-ions in both linear and nonlinear gel gradients. This is made possible by combining Ferguson's relationship for electrophoretic mobility versus gel concentration with the equation describing the gel gradient. Solution of these two equations yields the instantaneous velocity and position for each band as a function of time. This analysis provides insight into situations in which pore gradients may improve resolution and conditions for optimal resolution of multicomponent systems (macromolecular mapping). Position and instantaneous velocity of migration for any macromolecule on gel concentration (“pore”) gradient electrophoresis may be calculated for a linear gradient: χ= log e(ba 2u ot + exp (bT o)) − bT o ba 2 (iva) ν= u o (ba 2u ot + exp (bT o)) (va) This makes it possible to calibrate or standardize pore gradient electrophoresis. This analysis indicates that the proteins do not come to a “pore limit” or “dead stop.” Pore gradient electrophoresis is not indicated for analysis or separation of one, two, or three components, nor for charge fractionation. It is useful for simultaneous analysis of components of multicomponent mixtures (macromolecular mapping); and a “transverse” gradient presents a promising approach to obtaining Ferguson plots.