Ferguson Plot 

Measuring the electrophoretic mobility of a macromolecule at several gel (polymer) concentrations  provides information about its size and surface net charge density. In the ideal case, a plot of the logarithm of the mobility versus the gel concentration is linear and known as a Ferguson plot . The slope of such a plot gives the retardation coefficient, which is a measure of the size, shape, and conformation of the macromolecule. The intercept at zero gel concentration provides a measure of the free electrophoretic mobility, the mobility it would have in the absence of gel, which is related to the net charge on the molecular surface. In contrast, the mere inspection of one electrophoretic pattern or measurement of the mobility of one band gives no information whatsoever concerning the size or net charge of the migrating species; even a comparison with standard macromolecules is usually unreliable.

To generate a Ferguson plot, several gel (or polymer) concentrations are used simultaneously in a multitube or multichannel electrophoresis apparatus. Alternatively, a transverse gradient of gel concentration can be used in a single-slab gel. The slopes and intercepts of the Ferguson plot are computed and translated to parameters descriptive of the size and net charge, including their statistical limits, using relevant computer programs (1). Even without such translation, the Ferguson plot can be used to recognize qualitative relationships between the size or net charge of related bands of an electropherogram, or to test the possibility that two species are identical. Recognition of the nature of the relationships between bands helps to decide whether to use separation methods based primarily on size  or charge. A linear Ferguson plot is expected from a simple mathematical model of a rigid ball passing through an inert random fiber network of the gel matrix (the Ogston model); it is observed experimentally with relatively rigid and spherical proteins passing through gels electrophoretically. In contrast, Ferguson plots derived with agarose gels are convex, presumably due to the progressive supercoiling of double-helical agarose with decreasing concentration. They are concave when used with DNA fragments, presumably due to the progressive stretching of the DNA molecule with increasing gel concentration.

References

1. D. Tietz (1988) Adv. Electrophoresis 2, 109–170.