A Fluorescence-based Assay for Measuring Phospholipid Scramblase Activity in Giant Unilamellar Vesicles

Research output: Contribution to journal › Journal article › Research › peer-review

Standard

A Fluorescence-based Assay for Measuring Phospholipid Scramblase Activity in Giant Unilamellar Vesicles. / Mathiassen, Patricia P. M.; Pomorski, Thomas Gunther.

In: Bio-protocol, Vol. 12, No. 6, 4366, 2022.

Research output: Contribution to journal › Journal article › Research › peer-review

Harvard

Mathiassen, PPM & Pomorski, TG 2022, 'A Fluorescence-based Assay for Measuring Phospholipid Scramblase Activity in Giant Unilamellar Vesicles', Bio-protocol, vol. 12, no. 6, 4366. https://doi.org/10.21769/BioProtoc.4366

APA

Mathiassen, P. P. M., & Pomorski, T. G. (2022). A Fluorescence-based Assay for Measuring Phospholipid Scramblase Activity in Giant Unilamellar Vesicles. Bio-protocol, 12(6), [4366]. https://doi.org/10.21769/BioProtoc.4366

Vancouver

Mathiassen PPM, Pomorski TG. A Fluorescence-based Assay for Measuring Phospholipid Scramblase Activity in Giant Unilamellar Vesicles. Bio-protocol. 2022;12(6). 4366. https://doi.org/10.21769/BioProtoc.4366

Author

Mathiassen, Patricia P. M. ; Pomorski, Thomas Gunther. / A Fluorescence-based Assay for Measuring Phospholipid Scramblase Activity in Giant Unilamellar Vesicles. In: Bio-protocol. 2022 ; Vol. 12, No. 6.

Bibtex

@article{535c0d3f8c784fd1b7c0a5e2fb9f773d,
title = "A Fluorescence-based Assay for Measuring Phospholipid Scramblase Activity in Giant Unilamellar Vesicles",
abstract = "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.",
keywords = "ATTO488, Electroformation, Dithionite, Fluorescence microscopy, Giant unilamellar vesicle, Phospholipid scramblase, Fluorescence bleaching, ENDOPLASMIC-RETICULUM MEMBRANE, FLIP-FLOP, PHOSPHATIDYLSERINE, ELECTROFORMATION, RECONSTITUTION, MECHANISMS, PROTEINS, EXPOSURE",
author = "Mathiassen, {Patricia P. M.} and Pomorski, {Thomas Gunther}",
year = "2022",
doi = "10.21769/BioProtoc.4366",
language = "English",
volume = "12",
journal = "Bio-protocol",
issn = "2331-8325",
publisher = "bio-protocol",
number = "6",

}

RIS

TY - JOUR

T1 - A Fluorescence-based Assay for Measuring Phospholipid Scramblase Activity in Giant Unilamellar Vesicles

AU - Mathiassen, Patricia P. M.

AU - Pomorski, Thomas Gunther

PY - 2022

Y1 - 2022

N2 - 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.

AB - 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.

KW - ATTO488

KW - Electroformation

KW - Dithionite

KW - Fluorescence microscopy

KW - Giant unilamellar vesicle

KW - Phospholipid scramblase

KW - Fluorescence bleaching

KW - ENDOPLASMIC-RETICULUM MEMBRANE

KW - FLIP-FLOP

KW - PHOSPHATIDYLSERINE

KW - ELECTROFORMATION

KW - RECONSTITUTION

KW - MECHANISMS

KW - PROTEINS

KW - EXPOSURE

U2 - 10.21769/BioProtoc.4366

DO - 10.21769/BioProtoc.4366

M3 - Journal article

C2 - 35434199

VL - 12

JO - Bio-protocol

JF - Bio-protocol

SN - 2331-8325

IS - 6

M1 - 4366

ER -

ID: 304158700