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10 protocols using palmitoyl oleoyl phosphatidylcholine

1

Lipid Procurement for Biophysical Studies

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Dipalmitoyl-phosphatidylcholine (DPPC), palmitoyl-oleoyl-phosphatidylcholine (POPC), palmitoyl-arachidonoyl-phosphatidylcholine (PAPC), pamitoyl-docosahexaenoyl-phosphatidylcholine (PDPC), palmitoyl-oleoyl-phosphatidylethanolamine (POPE), palmitoyl-arachidonoyl-phosphatidylethanolamine (PAPE), palmitoyl-docosahexaenoyl-phosphatidylethanolamine (PDPE), stearoyl-docosahexaenoyl-phosphatidic acid (SDPA), egg phosphatidic acid (PA), and egg phosphatidylglycerol (PG) were obtained from Avanti Polar Lipids (Alabaster, Alabama) (S1 Table).
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2

Lipid Bilayer Incorporation of Dengue Virus M Protein

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Example 54

Lipid bilayer studies were performed as described elsewhere (Sunstrom, 1996; Miller, 1986). A lipid mixture of palmitoyl-oleoyl-phosphatidylethanolamine, palmitoyl-oleoyl-phosphatidylserine and palmitoyl-oleoyl-phosphatidylcholine (5:3:2) (Avanti Polar Lipids, Alabaster, Ala.) was used. The lipid mixture was painted onto an aperture of 150-200 μm in the wall of a 1 ml delrin cup. The aperture separates two chambers, cis and trans, both containing salt solutions at different concentrations. The cis chamber was connected to ground and the trans chamber to the input of an Axopatch 200 amplifier. Normally the cis chamber contained 500 mM KCl and the trans 50 mM KCl. The bilayer formation was monitored electrically by the amplitude of the current pulse generated by a current ramp. The potentials were measured in the trans chamber with respect to the cis. The protein was added to the cis chamber and stirred until channel activity was seen. The currents were filtered at 1000 Hz, digitized at 5000 Hz and stored on magnetic disk.

The dengue virus M protein C-terminal peptide (DMVC) was dissolved in 2,2,2-trifluoroethanol (TFE) at 0.05 mg/ml to 1 mg/ml. 10 μl of this was added to the cis chamber of the bilayer which was stirred. Channel activity was seen within 15-30 min.

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3

Lipid Extraction and Purification

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DPPC, palmitoyl-oleoyl phosphatidylcholine (POPC), and POPG were obtained from Avanti Polar Lipids (Alabaster, AL, USA) at >99% purity.
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4

Peptide and Lipid Synthesis for Biomembrane Studies

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ΔM4 (NFFKRIRRAWKRIWKWIYSA, Lot. b88771380001/PE2074) and LTX-315 (KKWWKKWDipK–NH2, Lot. 91799130007/PE4961) were purchased through a custom peptide synthesis service from GenScript (Piscataway Township, NJ, USA). The solvent removal was performed, the peptide purity was determined by analytical HPLC (higher than 95%), and the molecular weight was confirmed with MALDI-TOF mass spectrometry. Dipalmitoylphosphatidylcholine (DPPC, Lot. 160PC-318), sphingomyelin egg chicken (SM, Lot. 860061P-25MG-A-116), dipalmitoylphosphatidylethanolamine (DPPE, Lot. 160PE-106), dipalmitoylphosphatidylserine sodium salt (DPPS, Lot. 840037P-500MG-A-078CL750332P-200MG-A-030), palmitoyloleoylphosphatidylcholine (POPC, Lot. 850457P-500MG-A-211), palmitoyloleoylphosphatidylethanolamine (POPE, Lot. 850757P-500MG-B-151), and palmitoyloleoylphosphatidylserine sodium salt (POPS, Lot. 840034P-25MG-A-250) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). HEPES, NaCl, and EDTA were purchased from Sigma-Aldrich (St. Louis, MO, USA).
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5

Lipid Vesicle Preparation by Extrusion

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The appropriate volume of lipid stocks dissolved in chloroform were dried under a nitrogen stream and dried overnight using high vacuum. The dried lipids were re-suspended in 50 mM phosphate buffer, pH 8.0 to a final concentration of 20 mM and large unilamellar vesicles (LUV) were formed by extrusion using a Mini-Extruder (Avanti Polar Lipids, Alabaster, AL). Extrusion was performed using 0.1 μm nucleopore polycarbonate membranes (Whatman, Philadelphia, PA) and the prepared stocks were stored at −4 °C. Lipids used in this study: Palmitoyl-oleoyl-phosphatidylcholine (POPC), palmitoyl-oleoyl-phosphatidylserine (POPS), and 1-palmitoyol-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) were purchased from Avanti Polar Lipids (Alabaster, AL)
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6

Lipid Sourcing and Preparation

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Dilauroyl phosphatidylcholine (DLPC), dilauroyl phosphatidylglycerol (DLPG), palmitoyl oleoyl phosphatidylcholine (POPC), and palmitoyl oleoyl phosphatidylglycerol (POPG) were purchased from Avanti® Polar Lipids, Inc. Monosodium phosphate and disodium phosphate were purchased from Sigma-Aldrich.
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7

Reconstitution of diphtheria toxin

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Palmitoyl-oleoyl-phosphatidylcholine (POPC) and palmitoyl-oleoyl-phosphatidylglycerol (POPG) were purchased from Avanti Polar Lipids (Alabaster, AL). AlexaFluor488-maleimide was obtained from Invitrogen (Carlsbad, CA). Diphtheria toxin T-domain (amino acids 202–378) was cloned into NdeI-EcoRI-treated pET15b vector containing an N-terminal 6xHis-tag (Rodnin et al., 2008 (link)).
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8

Antimicrobial Peptide Lipid Interactions

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Aurein 1.1 (GLFDIIKKIAESF-NH 2 ), maculatin 1.1 (GLFGVLAKVAAHVVPAIAEHF-NH 2 ) and caerin 1.1 (GLLSVLGSVAKHVLPHVVPVIAEHL-NH 2 ) were synthesized by solid phase peptide synthesis and HPLC purified in house (Bio21 Institute, Dr J. Karas) at a purity >95%. Palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylglycerol (POPG), tetraoeloylcardiolipin (TOCL) and sphingomyelin from porcine brain (SM) phospholipids were purchased from Avanti Polar Lipids (Alabaster, USA) and used without further purification. Cholesterol (Chol), 5(6)-carboxyfluorescein (CF), Triton X-100 and PD-10 columns were purchased from Sigma (St Louis, USA).
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9

Lipid Nanoparticle Formulation Development

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Palmitoyl oleoyl phosphatidylcholine (POPC), dioleoylphosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), d18:1/18:0 C18 Lactosyl(ß) Ceramide (LAC), cholesterol, and egg sphingomyelin were obtained from Avanti Polar Lipids (Alabaster, AL). Astragaloside IV (AGIV) was obtained from the United States Pharmacopeial Convention (Rockville, MD). Other chemicals and solvents were obtained from Thermo Fisher (Waltham, MA) and Millipore Sigma (St. Louis, MO).
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10

Lipid Membrane Fusion Protocol

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Palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidyl-gycerol (POPG), palmitoyloleoylphosphatidylserine (POPS) were obtained from Avanti Polar Lipids (Alabaster, AL). Bovine thrombin was from Fisher Scientific (Pittsburgh, PA). The CLY3 construct containing the fusion of enhanced cyan and enhanced yellow fluorescent proteins was provided by Addgene (Cambridge, MA)[16 (link)]. The mutations were introduced using Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA) according to manufacturer’s protocol. 8-aminonaphtalene-1,3,6-trisulfonic acid, disodium salt (ANTS) and p-xylene-bis-pyridinium bromide (DPX) were obtained from Molecular Probes (Eugene, OR).
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