Reconstitution of Vesicle Budding from the TGN and Immunoisolation of Vesicles Enriched with a Specific Cargo Client

The trans-Golgi network (TGN) is an important cargo sorting station in the secretory pathway. To reveal insights into protein sorting at the TGN, it is important to develop an assay to quantify the efficiency of cargo capture. Here, we describe an experimental approach to reconstitute the packaging of cargo proteins into vesicles at the TGN in vitro. We also describe an experimental approach to immunoisolate vesicles enriched with a specific transmembrane cargo client from the in vitro vesicle formation assay. These assays provide robust tools to directly measure the enrichment of cargo proteins into TGN-derived vesicles and to reveal novel factors that regulate TGN export process.