Chapter 15 Immunoisolation Using Magnetic Solid Supports: Subcellular Fractionation for Cell-Free Functional Studies

Abstract

Publisher Summary In the endocytic pathway, the different steps of membrane traffic remain poorly understood and controversial. The experimental strategy to study membrane traffic in endocytosis has been to reconstitute a specific step of the pathway in a cell-free system using defined subcellular fractions. This chapter develops immunoisolation that uses the specificity of antibodies, as a subcellular fractionation method. The chapter uses monodisperse magnetic solid supports that efficiently and rapidly isolate the fraction of interest. The chapter describes the methods used in experimental approach. How immunoisolated fractions are used in assays of vesicle fusion are also described in the chapter. Immunoisolated fractions were used to study the in vitro reconstitution of endocytic-vesicle fusion. These fractions correspond to different times of G protein internalization. The G molecules move from the plasma membrane to an “early” tubulovesicular endosome located at the cell periphery and then to “late” acid phosphatase-positive endosomal structures with a complex morphology in the perinuclear region.