Purification of Plasma Membrane from Rice Leaves and Analysis of the Proteins by Two-Dimensional Electrophoresis

An effective method for the isolation of plasma membrane (PM) from rice,Oryza sativa,seedlings was established using aqueous two-phase system which was composed of Dextran T 500 and PEG 3350. The effect of the concentration of the polymer on partition of PM in polymer system was studied．Phase separation was carried out in a series of two-phase systems containing 6.1%-6.5% (w) of each polymer dissolved in phase buffer. The result indicated that the most effective phase partition system which consisted of 6.3%/6.3%(w) PEG 3350/Dextran T 500 in 0.25 mol/L sucrose, 5 mmol/L potassium phosphate and 3 mmol/L KCl (pH 7.8) was suitable for the isolation of PM from rice. The electron micrograph stained with uranyl actate-lead citrate stain and marker enzyme activities analysis proved that the isolated PM had obtained high purity (93.5%) and right-side out sealed vesicles. Purified PM proteins were also obtained from crude PM in good yield (2.73%). To improve the solubilization of hydrophobic PM proteins and dissociate protein complexes, PM proteins were treated by an optimized rehydration buffer including new zwitteronic detergents, just as ASB-14 and CHAPS. 1-D SDS-PAGE of purified PM proteins showed more band numbers and well-proportioned bands intensity than that of crude PM. PM proteins were also separated by IEF/SDS-PAGE. 579±17 well-resolved proteins with a much clearer background were obtained, which also showed good reproducibility among three independent experiments. The result above showed that the method to purify PM proteins using the aqueous two-phase partition system could be suitable for high-throughput PM proteomic analysis, such as SDS-PAGE combined with HPLC-ESI-MS/MS and 2-DE analysis.