Chapter 3 - Immunoisolation of Organelles Using Magnetic Solid Supports

Abstract

This chapter describes immuneisolation of organelles using magnetic solid supports. To set up an immunoisolation protocol, one first has to identify an appropriate antigen that is tightly associated with the membrane and localizes exclusively to the compartment that one wishes to isolate. It is possible to use either an indirect or a direct immunoisolation technique. In the first case, the antibody is bound to the organelle, and the complex is retrieved with a linker antibody that is bound to the solid support. In the second case, the antibody is first bound to a linker antibody that is bound to the solid support. Immunoisolated fractions can be analyzed by virtually any means, as the beads themselves are inert. Most types of analysis, including Western blotting, bideminsional gel electrophoresis, lipid analysis, electron microscopy, or enzymatic assays, can be performed using standard protocols. Before proceeding to immunoisolating the compartment of choice, a pre-purification step can be useful. A simple method for partially purifying organelles is the density centrifugation.