Polystyrene latex particles as size standards in quantitative agarose gel electrophoresis: Application to three plant viruses

Abstract

AbstractNegatively charged polystyrene latex particles with electron microscopically determined radii of 22.4, 26.9, 27.6, 43.5, 44.2 and 59.4 nm (nominal radii provided by the manufacturer of 28.5 to 75 nm) were found to be applicable as size standards for the quantitative agarose gel electrophoresis of viruses and cellular particles in that size range. In electrophoresis at pH 6.5, 0.03 M ionic strength in presence of 10 mM 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulfonate (CHAPS), the polystyrene particles exhibit non‐linear, reproducibly convex‐sigmoidal Ferguson plots. This curve shape is compatible with a model specifying a dependence of gel fiber dimensions and of particle size on the gel concentration. Based on a standardization of the agarose fiber by the polystyrene latex particles, computer simulation methods were used to determine the effective radii (without added Ca++) of turnip crinkle virus (TCV), hibiscus chlorotic ringspot virus (HCRSV) and pelargonium flowerbreak virus (PFBV) as 29.4, 24.2 and 22.1 nm, and their net charges as free mobilities, μo of 9.89, 10.87 and 16.20 · 10−5 cm2/s/V, respectively. Within the size range of the standards, a simplified procedure for determination of particle size from curved Ferguson plots appears applicable as a first approximation (Zwaan method). When electrophoresis was conducted in the presence of 5 mM Ca++, the effective particle radii of TCV, HCRSV and PFBV were reduced to 12.8, 20.3 and 18.3 nm, respectively. This compares with previous electron microscopic estimates of approximately 15 nm for TCV, HCRSV and PFBV. The observed increase of effective particle radii in the absence of Ca++ corroborates the particle swelling and/or shape change at pH's ≥ 5.5 in the absence of Ca++ which is known to occur with other plant viruses.