A Fluorescence-based Assay for Measuring Phospholipid Scramblase Activity in Giant Unilamellar Vesicles
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Transbilayer movement of phospholipids in biological membranes is mediated by a diverse set of lipid transporters. Among them are scramblases that facilitate rapid bi-directional movement of lipids without metabolic energy input. In this protocol, we describe the incorporation of phospholipid scramblases into giant unilamellar vesicles (GUVs) formed from scramblase-containing large unilamellar vesicles by electroformation. We also describe how to analyze their activity using membrane-impermeant sodium dithionite, to bleach symmetrically incorporated fluorescent ATT0488-conjugated phospholipids. The fluorescence-based readout allows single vesicle tracking for a large number of settled/immobilized GUVs, and provides a well-defined experimental setup to directly characterize these lipid transporters at the molecular level.
Originalsprog | Engelsk |
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Artikelnummer | 4366 |
Tidsskrift | Bio-protocol |
Vol/bind | 12 |
Udgave nummer | 6 |
Antal sider | 19 |
ISSN | 2331-8325 |
DOI | |
Status | Udgivet - 2022 |
Links
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8983165/pdf/BioProtoc-12-06-4366.pdf
Forlagets udgivne version
ID: 304158700