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2 protocols using 1 palmitoyl 2 oleoyl sn glycero 3 phosphatidylglycerol

1

Preparation of Lipid Vesicles for Biophysical Studies

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Lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (POPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), were obtained from Avanti Polar Lipids (Alabaster, AL). For circular dichroism, fluorescence and 1D NMR experiments, large unilamellar vesicles were prepared using extrusion as previously described [29 (link),30 (link)]. Vesicles with zwitterionic lipids, POPC (100%) and POPE/POPC (40/60 mol%), and with negatively charged lipids, POPG/POPC (40/60 mol%) and POPA/POPC (10/90, 20/80, 30/70, 40/60 and 50/50 mol%), were produced. Lipids were dissolved in chloroform and the chloroform was evaporated overnight under nitrogen gas. The lipid mixtures were resuspended in 1 ml of the appropriate buffer for the experiment for 30 min by vortexing the samples. Next, each sample went through five cycles of freezing in liquid nitrogen and thawing in a water bath (∼60 °C). Finally, samples were extruded through a 100 nm polycarbonate membrane (Avanti Polar Lipids, Alabaster, AL). The size of the extruded vesicles was analyzed by dynamic light scattering (ALV-laser, Langen, Germany) and vesicles were found to have a radius of around 65 nm.
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2

Peptide Synthesis and Lipid Preparation

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Peptide synthesis reagents and Fmoc-protected amino acids were purchased from Merck Biosciences (Darmstadt, Germany) and/or Iris Biotech (Marktretwitz, Germany). 15N-labelled amino acids were purchased from Cambridge Isotope Laboratories (Andover, MA, USA) and were Fmoc-protected using Fmoc-Cl as described previously69 . Solvents for peptide synthesis were purchased from Merck (Darmstadt, Germany) or from Biosolve (Valkenswaard, Netherlands) and solvents for HPLC purification were obtained from Fischer Scientific (Geel, Belgium). The lipids 1,2-dierucoyl-sn-glycero-3-phosphatidylcholine (DErPC), 1,2-dierucoyl-sn-glycero-3-phosphatidylglycerol (DErPG), 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPC), 1,2-dimyristoleoyl-sn-glycero-3-phosphatidylcholine (DMoPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhod-PE) were obtained from Avanti Polar Lipids (Alabaster, USA). The fluorescent probes 8-amino-naphtalene-1,3,6-trisulfonic acid sodium salt (ANTS) and pxylenebis(pyridinium)bromide (DPX) were obtained from Invitrogen - Molecular Probes (Karlsruhe, Germany).
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