Fluorescence Leakage Assay to Study Cell-Penetrating Peptide Interaction with Membrane

Published: April 30, 2023

Abstract

Source: Konate, K. et al. Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide. J. Vis. Exp. (2020)

This video discusses the fluorescence leakage assay to study the interactions of cell-penetrating peptides with cell membranes using cell membrane mimics — large unilamellar vesicles, or LUVs, encapsulated with a fluorescent dye and a quencher.

Protocol

1. Preparation of Large Unilamellar Vesicles (LUVs) Prepare LUVs for their use as cell membrane mimics for fluorescence leakage assay. Mix with a Hamilton glass syringe phosphatidylcholine (DOPC, 786.11 g/mol), sphingomyelin (SM, 760.22 g/mol) and Cholesterol (Chol, 386.65 g/mol) at the molar ratio 4:4:2. The lipid solution is obtained from a stock solution of each lipid solubilized in a methanol/chloroform (3/1; volume/volume) solvent at 25 mg/mL in a 25 mL glass round-bottom flask. Based on 4 &#18…

Açıklamalar

The authors have nothing to disclose.

Materials

25 mL glass round-bottom flask Pyrex
8-aminonaphthalene-1, 3, 6-trisulfonic acid, disodium salt (ANTS) Invitrogen A350 Protect from light
Chloroform Sigma-Aldrich 288306
Cholesterol Sigma-Aldrich C8667
DOPC (dioleoylphosphatidylcholine) Avanti Polar 850375P Protect from air
Extruder Avanti Polar 610000
Fluorimeter PTI Serlabo
50 µL glass syringe Hamilton 705N
HEPES Sigma-Aldrich H3375
LabAssay Phospholipid WAKO 296-63801
liquid chromatography column Sigma-Aldrich
Methanol Carlo Erba 414902
Nuclepore polycarbonate membrane (0.1 µm pore size, 25 mm diameter) Whatman 800309
polystyrene cuvette, 10 x 10 x 45 mm Grener Bio-One 614101
polystyrene semi-micro cuvette, DLS Fisher Scientific FB55924
p-xylene-bispyridinium bromide (DPX) Invitrogen X1525 Protect from light
quartz fluorescence cuvette Hellma 109.004F-QS
rotavapor system Heidolph Z334898
Sephadex G-50 resin Amersham 17-0042-01
Sodium azide (NaN3) Sigma-Aldrich S2002
Sodium chlorid (NaCl) Sigma-Aldrich S5886
Sonicator bath USC300T VWR 142-6001
Sphingomyelin Avanti Polar 860062P Protect from air
Triton X-100 Eromedex 2000-B
Zetaziser NanoZS Malvern ZEN3500

Etiketler

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Bu Makaleden Alıntı Yapın
Fluorescence Leakage Assay to Study Cell-Penetrating Peptide Interaction with Membrane. J. Vis. Exp. (Pending Publication), e21272, doi: (2023).

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