Abstract
Cell-derived vesicles facilitate the isolation of transmembrane proteins in their physiological membrane maintaining their structural and functional integrity. These vesicles can be generated from different cellular organelles producing, housing, or transporting the proteins. Combined with single molecule imaging, isolated organelle specific vesicles can be employed to study the trafficking and assembly of the embedded proteins. Here we present a method for organelle specific single molecule imaging via isolation of ER and plasma membrane vesicles from HEK293T cells by employing OptiPrep gradients and nitrogen cavitation. The isolation was validated through Western blotting, and the isolated vesicles were used to perform single molecule studies of oligomeric receptor assembly.
Document Type
Article
Publication Date
5-5-2018
Digital Object Identifier (DOI)
https://doi.org/10.21769/BioProtoc.2825
Funding Information
This work was supported in part by the NIDA training grant DA016176 (AMF) and NIDA DA038817.
Repository Citation
Moonschi, Faruk H.; Fox-Loe, Ashley M.; Fu, Xu; and Richards, Christopher I., "Mammalian Cell-Derived Vesicles for the Isolation of Organelle Specific Transmembrane Proteins to Conduct Single Molecule Studies" (2018). Chemistry Faculty Publications. 149.
https://uknowledge.uky.edu/chemistry_facpub/149
Notes/Citation Information
Published in Bio-protocol, v. 8, issue 9, p. 1-10.
Copyright © 2018 The Authors; exclusive licensee Bio-protocol LLC.
The publisher has granted the permission for posting the article here.