Determination of molecular weights and Stokes' radii of non‐denatured proteins by polyacrylamide gradient gel electrophoresis. 1. An equation relating total polymer concentration, the molecular weight of proteins in the range of 104–106, and duration of electrophoresis

Abstract

AbstractUntreated and processed gel plates of polyacrylamide (PAA) gradient gels were cut into strips perpendicularly to their length, and the wet and dry matter of the sections was determined. In untreated gels the apparent dry matter, as well as the relative dry matter, are a linear function of the gel length. In processed gels, however, only the apparent gel concentration increases linearly with the gel length, whereas the relative dry matter increases linearly with the square root of the gel length. The %T (content in polyacrylamide) was calculated from the apparent dry matter. The gel gradients used were found to be linear with respect to %T. Six different calibration proteins were run and their time‐dependent migration distances determined. For each protein a linear relationship exists between the square root of its migration distance (√D) and the logarithm of the electrophoretical duration (log t). In this relation √D can be substituted by the square root of the PAA concentration \documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt {\%{\rm T}} $\end{document} or by the relative dry matter (TM(%)). The resulting straight lines seem to intersect near the zero time of electrophoresis and the starting concentration of the gradient. At any time during electrophoresis (t» O), the square root of the migration distance (√D) of the calibration proteins and the logs of their Stokes' radii (log Rs), or the logs of their molecular weights (log MW), are correlated linearly. In this function, √D may be replaced by either \documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt {\%{\rm T}} $\end{document} or TM (%). It is shown that this correlation is not subject to time, although the constants are changed during the process of electrophoresis. The slopes of the lines and the duration of the electrophoresis (at a constant voltage) are related by a hyperbolic function. Regression lines obtained for different times during electrophoresis intersect in the range of the starting gel concentration. A more general equation is formulated which states ‐ within certain experimental limitations — the size of proteins, their migration distances, and the time of electrophoresis.