Analysis of convex Ferguson plots in agarose gel electrophoresis by empirical computer modeling

Abstract

Agarose gel electrophoresis has been shown to give rise to non‐linear plots of log (mobility) vs. gel concentration of spherical viruses (Serwer,[1]) and cellular vesicles (Gottlieb et al.[2]). This finding also applies to proteins as shown in this study. Considering that in the non‐linear plot, the slope becomes a function of gel concentration, it is possible to determine particle properties and gel parameters by a modification of the conventional method derived from the Ogston theory for long‐fiber gels. This treatment shows: (a) In application to data obtained from gel electrophoresis (0.4 to 1.6 % agarose) of viruses (13 to 42 nm radius) and with increasing gel concentration: (i) an increase of apparent total fiber length per g agarose matrix, (ii) a reduction of apparent fiber radius and (iii) a constant fiber volume (per g matrix material) of the agarose fiber. The values of the fiber radii identify the fibers as agarose supercoils (or aggregates of it) with a radius of 20–55 nm. (b) In application to data obtained from gel electrophoresis (1.2 to 8 % agarose) of proteins (1.7 to 5.8 nm radius): a fiber volume indicative of additional sieving by the agarose double helix (of known radius of 0.5–2 nm). This is in agreement with a previous suggestion by Serwer [1] that proteins are able to penetrate into the double‐helical network of which the agarose supercoil consists. (c) The likelihood of a continuous transition from case (a) to case (b).

Stacking of unlabeled sodium dodecyl sulfate-proteins within a fluorimetrically detected moving boundary, electroelution and mass spectrometric identification

“…(iii) Consistent with the widening of a stack in proportion to load of constituents [11][12][13], the fluorescently detected band increases in area with increasing load of proteins A, B and C (Fig. 3) and with increased SDS-load from 1 to 5% in the sample, the latter resulting in a 2.4-fold increase in band area.…”

“…(iv) Consistently with the unstacking of constituents when their net mobility is lowered below that of the trailing constituent, either by raising the gel concentration or by increasing the size of the constituent, it was found that SDS-proteins A, C and E comigrate at agarose concentrations below 4%, A and C comigrate (at reduced mobi- The initial and final slopes of the convex Ferguson plots [12] are designated KR-I and KR-II, respectively. Hypothetically, proteins A and C are stacked at all gel concentrations, while protein E is unstacked above 4% agarose concentration.…”

Electrophoresis in Polymer Solutions: Mechanisms of Molecular Sieving

“…The program SigmaPlot (Jandel Scientific) was used for curve fitting on the Macintosh computer. Figure 1 shows a representative example (phycoerythrin, R ) 4.0 nm) for the relation between the local retardation coefficient [K R ′ ) log(µ/µ 0 )/c] 18 and PEG molecular weight at a PEG concentration of 0.75%. The transition between the dilute and semidilute regimes at 0.75% PEG is shown by the arrow.…”

Reproducibility of mobility in gel electrophoresis

“…Clearly, these provisos do not apply to DNA fragments, which are flexible, and do depend in their structures on field strength and gel concentration [ 11 and therefore require a future determination as to the confidence limits of their electrophoretic mobilities. Similarly, the degree of reproducibility of mobilities remains unknown for agarose gel electrophoresis at low concentrations, at which the transition from double-helical to supercoiled gel structure takes place [19].…”