Abstract
This chapter discusses the expression, purification, and characterization of Rab5 effector complex, Rabaptin-5/Rabex-5. The purification of the native complex is performed from bovine brain cytosol. For this purpose, four fresh brains are obtained from the slaughterhouse and quickly put on ice to avoid protein degradation. Further steps involve; homogenization, precipitation, gel filtration, ion exchange, and metal ion chromatography. To evaluate the purified Rabaptin-5 complex, one can use an in vitro homotypic endosome fusion assay. The assay consists of the incubation of biotinylated transferrinlabeled early endosomes with anti-transferrin antibody-labeled early endosomes in the presence of HeLa cytosol, an ATP-regenerating system, an unlabeled holotransferrin quencher, buffer, and water in a total volume of 20 μl. Measurements of fusion scores are taken for the content mixing between the endosomes containing antibody and its antigen, using streptavidin-coated magnetic beads and an Origen analyzer.
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