Cell-free Reconstitution of the Packaging of Cargo Proteins into Vesicles at the trans Golgi Network

Xiao Tang; Feng Yang; Yusong Guo

doi:10.21769/BioProtoc.3537

Cell-free Reconstitution of the Packaging of Cargo Proteins into Vesicles at the trans Golgi Network

Bio Protoc. 2020 Mar 5;10(5):e3537. doi: 10.21769/BioProtoc.3537.

Authors

Xiao Tang¹, Feng Yang¹, Yusong Guo¹

Affiliation

¹ Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, China.

Abstract

Protein sorting at the trans Golgi network (TGN) plays important roles in targeting newly synthesized proteins to their specific destinations. The aim of this proposal is to reconstitute the packaging of non-Golgi resident cargo proteins into vesicles at the TGN, utilizing rat liver cytosol, semi-intact mammalian cells and nucleotides. The protocol describes how to perform the vesicle formation assay, how to isolate vesicles and how to detect cargo proteins in vesicles. This reconstitution assay can be used to quantitatively measure the efficiency of the packaging of a specific cargo protein into transport vesicles at the TGN under specific experimental conditions.

Keywords: Budding; Cargo sorting; Reconstitution; Secretion; TGN; Vesicles.