A Fluorescence-based Assay for Measuring Phospholipid Scramblase Activity in Giant Unilamellar Vesicles
Research output: Contribution to journal › Journal article › Research › peer-review
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.
Original language | English |
---|---|
Article number | 4366 |
Journal | Bio-protocol |
Volume | 12 |
Issue number | 6 |
Number of pages | 19 |
ISSN | 2331-8325 |
DOIs | |
Publication status | Published - 2022 |
- ATTO488, Electroformation, Dithionite, Fluorescence microscopy, Giant unilamellar vesicle, Phospholipid scramblase, Fluorescence bleaching, ENDOPLASMIC-RETICULUM MEMBRANE, FLIP-FLOP, PHOSPHATIDYLSERINE, ELECTROFORMATION, RECONSTITUTION, MECHANISMS, PROTEINS, EXPOSURE
Research areas
Links
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8983165/pdf/BioProtoc-12-06-4366.pdf
Final published version
ID: 304158700