Journal
NATURE PROTOCOLS
Volume 8, Issue 6, Pages 1048-1057Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2013.061
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Funding
- Biotechnology and Biological Sciences Research Council (BBSRC)
- US National Science Foundation (NSF) [1253565]
- European Research Council (ERC)
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1253565] Funding Source: National Science Foundation
- BBSRC [BB/D010918/1, BB/E019668/1] Funding Source: UKRI
- EPSRC [EP/H02994X/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/D010918/1, BB/E019668/1] Funding Source: researchfish
- Engineering and Physical Sciences Research Council [EP/D001846/1, EP/H02994X/1] Funding Source: researchfish
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We describe a protocol for forming an artificial lipid bilayer by contacting nanoliter aqueous droplets in an oil solution in the presence of phospholipids. A lipid monolayer forms at each oil-water interface, and when two such monolayers touch, a bilayer is created. Droplet interface bilayers (DIBs) are a simple way to generate stable bilayers suitable for single-channel electrophysiology and optical imaging from a wide variety of preparations, ranging from purified proteins to reconstituted eukaryotic cell membrane fragments. Examples include purified proteins from the a-hemolysin pore from Staphylococcus aureus, the anthrax toxin pore and the 1.2-MDa mouse mechanosensitive channel MmPiezo1. Ion channels and ionotropic receptors can also be reconstituted from membrane fragments without further purification. We describe two approaches for forming DIBs. In one approach, a lipid bilayer is created between two aqueous droplets submerged in oil. In the other approach, a membrane is formed between an aqueous droplet and an agarose hydrogel, which allows imaging in addition to electrical recordings. The protocol takes <30 min, including droplet generation, monolayer assembly and bilayer formation. In addition to the main protocol, we also describe the preparation of Ag/AgCl electrodes and sample preparation.
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