A Fluorescence Dequenching-based Liposome Leakage Assay to Measure Membrane Permeabilization by Pore-forming Proteins

Javier Aguilera; Salvador Vazquez-Reyes; Jianjun Sun

doi:10.21769/BioProtoc.4025

A Fluorescence Dequenching-based Liposome Leakage Assay to Measure Membrane Permeabilization by Pore-forming Proteins

Bio Protoc. 2021 May 20;11(10):e4025. doi: 10.21769/BioProtoc.4025.

Authors

Javier Aguilera¹, Salvador Vazquez-Reyes¹, Jianjun Sun¹

Affiliation

¹ Department of Biological Sciences and Border Biomedical Research Center, 500 West University Avenue, University of Texas at El Paso, El Paso, TX 79968, USA.

Abstract

Pore-forming toxins (PFTs) have been discovered in a wide range of organisms. Their functions are essential to the survival or virulence of many species. PFTs often interact with lipid membranes. Large unilamellar vesicles (LUV), also known as liposomes, have been commonly used as reliable membrane models for testing PFTs activity. Liposomes have great adaptability in size, lipid composition, and loading cargo. Incorporating the fluorescent dye/quencher pair, 8-Aminonaphthalene-1,3,6-Trisulfonic Acid (ANTS) and p-Xylene-Bis-Pyridinium Bromide (DPX), in liposomes is an effective approach for measuring membrane leakage. When ANTS and DPX are encapsulated in a liposome, the fluorescence of ANTS is quenched by DPX. However, disruption of liposome integrity and subsequent leakage result in measurable fluorescence emitted by ANTS. Here, we report our protocol for optimal liposome preparation for measuring liposome leakage by fluorescence dequenching.

Keywords: ANTS; DPX; EsxA; EsxB; Fluorescence dequenching; Liposome leakage; Mycobacterium tuberculosis.

Abstract

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