A single vesicle-vesicle fusion assay for in vitro studies of snares and accessory proteins

Jiajie Diao, Yuji Ishitsuka, Hanki Lee, Chirlmin Joo, Zengliu Su, Salman Syed, Yeon Kyun Shin, Tae Young Yoon, Taekjip Ha

Research output: Contribution to journalArticle

Abstract

Snare(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. this protocol describes our fluorescence resonance energy transfer (Fret)-based single vesicle-vesicle fusion assays for snares and accessory proteins. Both lipid-mixing (with Fretpairs acting as lipophilic dyes in the membranes) and content-mixing assays (with Fretpairs present on a Dnahairpin that becomes linear via hybridization to a complementary Dna) are described. these assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. the details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. these assays can be used to study the roles of various snareproteins, accessory proteins and effects of different lipid compositions on specific fusion steps. the total time required to finish one round of this protocol is 3-6 d.

Original languageEnglish (US)
Pages (from-to)921-934
Number of pages14
JournalNature protocols
Volume7
Issue number5
DOIs
StatePublished - Nov 2012

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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    Diao, J., Ishitsuka, Y., Lee, H., Joo, C., Su, Z., Syed, S., Shin, Y. K., Yoon, T. Y., & Ha, T. (2012). A single vesicle-vesicle fusion assay for in vitro studies of snares and accessory proteins. Nature protocols, 7(5), 921-934. https://doi.org/10.1038/nprot.2012.020