Moving boundary electrophoresis on agarose gel of plant viruses and polystyrene microspheres

Abstract

AbstractThree plant viruses: turnip crinkle (TCV), hibiscus chlorotic ringspot (HCRSV) and pelargonium flowerbreak (PFBV), and polystyrene size standards with radii of 22.4–59.4 nm can be stacked within trailing and leading ion net mobilities of 0.059 to 0.273 (relative to Na+). Stacking was carried out at pH 6.50, 0.03 M ionic strength, 50 mM 3–[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulfonate, at a gel concentration of 0.4 %, in agarose gel electrophoresis conducted at 1.2 mA/cm2 of gel. Unstacking occurs under the same conditions at gel concentrations ranging from 0.5 to 1.1 % agarose, while it can be brought about between 0.1 and 0.7 % agarose when the pH is raised to 7.27, corresponding to a front moving boundary with a trailing ion net mobility of 0.216 (relative to Na+). Ferguson plots of viruses and polystyrene particles in the discontinuous buffer system are curvilinear and comparable to those obtained in a continuous buffer at pH 6.50 of the same composition and operative pH as that of the resolving phase of the discontinuous buffer. Particle radii and net charge values can be obtained from the non‐linear Ferguson plot in the discontinuous buffer system by previously reported methods of computer simulation, but this Ferguson plot presents a more limited data base than that in the continuous buffer since it excludes gel concentrations which yield relative mobility (Rf) values of 1.0. Since computer simulation provides the range of gel concentrations in which both the fiber radius and length [1], as well as the size of the particle [2], remain constant for a particular preparation of agarose, a simplified alternative method of evaluating particle sizes exists. Within that specific gel concentration range, the linear segment of the Ferguson plot can be used to compute particle sizes by an operationally convenient, albeit approximative, method, using the same PAGE‐PACK programs of D. Rodbard which are commonly used in the size determination of macromolecules by polyacrylamide gel electrophoresis. The two methods of particle size determination, based on either the entire non‐linear Ferguson plot or on its linear segment in the appropriate gel concentration range, yield similar results (average deviation 12 %). The radii of three plant viruses are dependent to different degrees on the presence of Ca++ in the electrophoretic system. Values obtained in the presence of Ca++ are comparable to those found by electron microscopy.