Ferguson plots

Butterman, M Tietz, D Orban, L Chrambach, A, Ferguson Plots Based on Absolute Mobilities in Polyarcylamide Gel Electrophoresis Dependence of Linearity of Polymerization Conditions and Application on the Determination of Free Mobility, Electrophoresis 9, 293, 1988. Caglio, S Chiari, M Righetti, PG, Gel Polymerization in Detergents Conversion Efficiency of Methylene Blue vs. Persulfate Catalysis, as Investigated by Capillary Zone Electrophoresis, Electrophoresis 15, 209, 1994. 

Chiari, M D Alesio, L Consonni, R Righetti, PG, New Types of Large-Pore Polyacrylamide-Agarose Mixed-Bed Matrices for DNA Electrophoresis Pore Size Estimation from Ferguson Plots of DNA Eragments, Electrophoresis 16, 1337, 1995. 

Figure 9.8 Ferguson plots for polyethylene oxide used as sieving polymer. The graphs represent plots of the polymer concentration and the log of electrophoretic mobility for six different samples. Graphs A, B, and C represent polymers with MW of 100, 300, and 900 kDa, respectively (From A. Guttman, Electrophoresis, 16 611 (1995). With permission.)...

Figure 9.2 Native gel electrophoresis of the Tetrahymena P4—P6 RNA. (A) The folded and extended forms equilibrate rapidly, producing a single band whose mobility reflects the average structure of the RNA. U1, U2, BP5/5a refer to mutations in a hinge region. RNAs were run on native 6% (19 1 monorbis) polyacrylamide gel in TBE + 10 mM MgCl2 at 25 C. (B) Ferguson plot shows that the relative mobility (M) depends linearly on gel concentration, as predicted by the Ogston model. Reprinted from Szewczak and Cech (1997).

The gel concentration required for polyacrylamide gel electrophoresis (PAGE) to achieve optimal resolution of two proteins (or nucleic acids) can be determined by measuring the relative mobility of each protein in a series of gels of different acrylamide concentrations to construct a Ferguson plot (log 10Rf versus %7)... 

The mobility of the solute molecules is an apparent logarithmic function of the molecular weight, usually resulting in linear so-called Ferguson plots [7] crossing each other at zero gel concentration. 

The mobility of a protein of unknown mass is compared with that of a series of proteins of broadly similar shape but known mass. The mobility of each protein (known and unknown) is determined at a series of acrylamide concentrations, and for each protein a plot of log Rf vs Cf is made (called a Ferguson plot), from whose slopes values of JCR are determined (Figure 4-26 ). In practice, five or more acrylamide concentrations would be needed for reliable results. A secondary plot of the derived values of KR against Mr for the standard proteins then enables the native molecular weight of the unknown protein to be determined. 

Figure 4-26. Ferguson analysis for determining the molecular weights of native proteins. The mobilities of the unknown and standard proteins are measured in the same buffer conditions but at various acrylamide/ bis-acrylamide concentrations (CT) at least five different concentrations should be used, and the ratio of acrylamide bis-acrylamide should be held constant. From the slopes of the Ferguson plots (log Rf vs. CT) for the various proteins (unknown and standards) the molecular weights of the unknown can be determined.

FIGURE 4. Ferguson plot of Lake Plussee DOC from 1 m depth, June 30, 1979 (from Muenster, 1982). FI and F2 are PAGE fractions of high and low electrophoretic mobility. BPS(IS) = Bromphenol Blue as the internal standard. 

Pressure-induced conformation changes in proteins can be studied using electrophoresis in multiple gel rods with different acrylamide concentrations and using Ferguson plots. 

Fig. 8.8 Polyacrylamide-gel electrophoresis of the tetrameric form (G4) of human butyrylcholinesterase in microcapillary gel tubes with different gel concentrations, T. Gel patterns for electrophoresis at 2 kbar and 20°C for (a) butyrylcholinesterase, and (b) butyrylcholinesterase in the presence of 2 m sorbitol (the gels were stained for enzyme activity according to the procedure of Karnovsky and Roots ). The numbers indicate the acrylamide concentrations T. (c) Ferguson plots constructed for butyrylcholinesterase at different pressures (o), atmospheric pressure, ( ) 0.5 kbar, ( ) 1 kbar, (A) 1.5 kbar, and ( ) 2 kbar. (d) A secondary plot of /Cr against pressure for ( ) bovine serum albumin, ( ) butyryicholinesterase, and ( ) butyrylchoiinesterase in the presence of 2 m sorbitol, (e) A secondary plot of log Vo against pressure for butyrylchoiinesterase.

Gel Concentration, Ferguson Plots and Molecular Weights of Native Proteins... 

To improve accuracy in measuring R, sieving in gels more concentrated than 0.9% (i.e., in the region of convex curvature in Ferguson plots for particles 13-42 nm in R) was measured by using... 

By extrapolating to zero field strength it is possible to find an inverse relationship between electrophoretic mobility and molecular weight (14). This relationship contrasts with that obtained with a Ferguson plot (15) normally used for proteins, but also frequently and often erroneously used for DNA. However, the inverse relationship is fully predicted by reptation theory (3, 9). In summary, determining precise DNA molecular weights using gel electrophoresis requires careful attention to experimental detail and Judicious choice of data workup. 

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