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 language | English |
|---|---|
| Pages (from-to) | 921-934 |
| Number of pages | 14 |
| Journal | Nature Protocols |
| Volume | 7 |
| Issue number | 5 |
| DOIs | |
| State | Published - Nov 2012 |
Bibliographical note
Funding Information:acknowleDGMents This work was supported by the US National Institutes of Health Grants (R21 GM074526 to T.H. and R01 GM51290 to Y.-K.S.) and by the National Research Foundation of Korea grants funded by the Korean government