Free flow zonal electrophoresis for fractionation of plant membrane compartments prior to proteomic analysis

Abstract

Free flow zonal electrophoresis (FFZE) is a versatile, reproducible, and potentially high-throughput technique for the separation of plant organelles and membranes by differences in membrane surface charge. It offers considerable benefits over traditional fractionation techniques, such as density gradient centrifugation and two-phase partitioning, as it is relatively fast, sample recovery is high, and the method provides unparalleled sample purity. It has been used to successfully purify chloroplasts and mitochondria from plants but also, to obtain highly pure fractions of plasma membrane, tonoplast, ER, Golgi, and thylakoid membranes. Application of the technique can significantly improve protein coverage in large-scale proteomics studies by decreasing sample complexity. Here, we describe the method for the fractionation of plant cellular membranes from leaves by FFZE.