Preparation of a PDMS Block to Form the Microfluidic Channel
3:17
Formation of the Tethered Supported Bilayer
6:18
Checking Bilayer Fluidity
7:06
Introducing v-SUVs into the Microfluidic Channels
7:59
Observing the Fusion of Single v-SUVs and the SBL
8:42
Results: Docking and Fusion Events Described by Lipid and Content Release
10:26
Conclusion
필기록
The overall goal of this assay is to observe individual membrane fusion events driven by neuronal and exocytotic SNARE proteins to understand fundamental mechanisms of neurotransmitter and hormone release. This method can help answer key questions
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Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.