Protein Ferguson plots

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 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.

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

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

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. 

In the second method for Mr measurements, this can be done by relying on a mathematical canceling of charge effects following measurements of mobility of native proteins in gels of different concentrations. This is the Ferguson plot described above ... 

Figure 3 Ferguson plot of bovine v-crystallin proteins. Four different gel concentrations (5.0%, 6.25%, 7.3%, 8.3% T) with Tris-glycine gel buffer were used for determination of the slope, Kr, of each subtraction, which is proportional to the relative molecular mass, M of the native protein. (Reprinted with permission from Chiou SH and Wu SH (1999) Evaluation of commonly used electrophoretic methods for the analysis of proteins and peptides and their application to biotechnology. Analytica Chimica Acta 383 47-60 Elsevier.)...

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