Purification of Plasma Membranes by Affinity Partitioning

This chapter describes the purification of rat liver plasma membranes by affinity partitioning in an aqueous polymer two-phase system using the lectin wheat germ agglutinin (WGA) as affinity ligand. Affinity partitioning is advantageous for conventional membrane fractionation techniques in being highly selective, allowing the rapid and high-yield purification of membranes. In addition, the aqueous polymer environment is gentle to membrane structure and function, which is of importance when studying labile structures and components.
A two-phase system will form when aqueous solutions of two structurally different polymers are mixed at sufficiently high concentrations (1). A commonly used polymer pair is polyethylene glycol 3350 (PEG) and Dextran T500. These polymers will form a two-phase system at concentrations above 5.4% (w/w) of each, the top phase being enriched in PEG and the bottom phase in Dextran. A conventional two-phase system like this may be used for the fractionation of biological material, including membranes. The distribution of material between the phases is modulated by altering polymer concentration and salt contents of the system (2), but the selectivity is often insufficient for the ready separation of membranes.