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1 palmitoyl 2 oleoyl sn glycero 3 phosphatidylcholine

Manufactured by Avanti Polar Lipids
Sourced in United States

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine is a synthetic phospholipid that is commonly used in biochemical research and applications. It is a key component of cell membranes and plays a role in various biological processes.

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15 protocols using 1 palmitoyl 2 oleoyl sn glycero 3 phosphatidylcholine

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

Lipid Synthesis and Characterization

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1-Palmitoyl-2-oleoyl-sn-glycero-3- phosphatidylcholine (POPC) and 1,2-dipalmitoyl-phosphatidylcholine (DPPC) were obtained from Avanti Polar Lipids (Alabaster AL). N-palmitoyl sphingomyelin (16:0 SM) and N-oleoyl sphingomyelin (18:1Δ9(c) SM) were produced by reacylation of lyso-SM with the desired fatty acyl residue and purified as described previously.13 and refs. 1-Palmitoyl-2-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-heptanoyl)-sn-glycero-3-phosphocholine (Me4-BODIPY-8-PC or B-PC) was synthesized and purified as described earlier17 (Figure 1A).
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3

Lipid Preparation and Characterization

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1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine
(POPC) and 1,2-dipalmitoylphosphatidylcholine (DPPC) were obtained
from Avanti Polar Lipids (Alabaster, AL). N-Palmitoylsphingomyelin
(16:0 SM) and N-oleoylsphingomyelin (18:1Δ9(c) SM) were produced by reacylation of lyso-SM with the
desired fatty acyl residue and purified as described previously (ref (13 (link)) and references therein).
1-Palmitoyl-2-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-heptanoyl)-sn-glycero-3-phosphocholine
(Me4-BODIPY-8-PC or B-PC) was synthesized and purified
as described earlier17 (Figure 1A).
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4

Lipid and Peptide Preparation for Biophysical Studies

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Lipids including 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol (Chol), and cardiolipin 1′,3′-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol (TOCL) are purchased from Avanti Polar Lipids (Alabaster, AL). 5-doxyl stearic acid (5-SASL), TmCl3·6H2O, and VC (vitamin C) are purchased from Sigma-Aldrich (St. Louis, MO). C-terminal amidated peptide PrP106-126 (K106TNMKHMAGAAAAGAVVGGLG126-NH2) is synthesized by the solid synthesis method. For simplicity, we will refer to the peptide as PrP106-126 without explicitly noting the amidation. Peptide purity is 98% as indicated by analytical HPLC elution. Fresh solutions containing monomeric PrP106-126 are prepared by dissolving lyophilized peptide in 10 mM HEPES pH 7.4. Centrifugation is used to remove preexisting insoluble aggregates.
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5

Reconstitution and Characterization of αABc Complex

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Cholesterol (Chol), sphingomyelin (SM), and phospholipids (PLs): 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylCholine (POPC), 1-palmditoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS), were obtained dissolved in chloroform from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Cholesterol analog cholestane spin label (CSL), HEPES, Tris-HCl, NaN3, sodium chloride (NaCl) lysozyme, deoxycholic acid, and DNase I were obtained from Sigma Aldrich (St. Louis, MO, USA). Isopropyl-1-thio-β-D-galactopyranoside (IPTG), ampicillin, phenylmethylsulfonyl fluoride, and polyethyleneimine were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Recombinant human αAc and αBc were expressed and purified, and the reconstituted 3:1 heteromeric complex of αAc to αBc (i.e., αABc) was prepared using the methods described in Section 4.2 and stored in HEPES buffer (10 mM HEPES, 100 mM NaCl, pH = 7.4). All preparations of α-crystallin (αAc, αBc, and αABc) and the Chol/MHLL membranes, as well as associated binding studies, were performed in HEPES buffer (10 mM HEPES, 100 mM NaCl, pH = 7.4).
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6

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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7

Lipid-Protein Interaction Study Protocol

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Cholesterol (Chol), egg sphingomyelin (SM), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). The cholesterol analog cholestane spin-label (CSL), bovine eye lens α-crystallin (C4163), HEPES, and sodium chloride (NaCl) were obtained from Sigma Aldrich (St. Louis, MO, USA). Bovine α-crystallin purchased from Sigma Aldrich was used without further purification. The average molecular weight of the α-crystallin subunit was determined to be 20.35 kDa based on the information αA = 19.8 kDa, αB = 22 kDa, and αA:αB = 3:1 from Sigma Aldrich.
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8

Lipid Membrane Composition Analysis

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Dichloromethane and methanol (HPLC-grade purity) were purchased from Merck (Darmstadt, Germany); cholesterol (chol), D2O (99% purity) and Phosphate Buffer Saline (PBS) tabs were purchased from Sigma Aldrich (St. Louis, MO, USA). 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-d31-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (d31-POPC), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0–22:6PC), 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (22:6–22:6PC), spin-labelled phosphatidylcholines (1-palmitoyl-2- stearoyl-(n-doxyl)-sn-glycero-3-phosphocholine, n-PCSL) with the nitroxide group in the positions 5, 7, 10 and 14 of the acyl chain, and 25-doxyl-cholesterol (CNO) were purchased from Avanti Polar Lipids (Birmingham, AL, USA). Molecular structures of the lipids used in this study are displayed in Figures S1 and S2 (Supporting Material).
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9

Lipid Composition for Membrane Studies

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1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), soy l-α-phosphatidylinositol (PI), 1-oleoyl-2-(6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionyl)amino)-hexanoyl)-sn-glycero-3-phosphoinositol (TopFluor TMR PI), 1-oleoyl-2-(6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionyl)amino)-hexanoyl)-sn-glycero-3-phosphatidylethanolamine (TopFluor TMR PE), and 1-palmitoyl-2-{12-[(7-nitro-2–1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphatidylcholine (NBD PC) were purchased from Avanti Polar Lipids (Alabaster, AL). Buffer components tris(hydroxymethyl)-aminomethane (Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium chloride, ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), and benzamidine were purchased from Fisher Scientific (Pittsburgh, PA). Dodecyl thiomaltopyranoside was purchased from Anatrace (Maumee, OH). The buffer solution used for all experiments contained 50 mM Tris, 600 mM HEPES, 300 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 4 mM benzamidine, and 24.7 μM Dodecyl thiomaltopyranoside (pH 8.0) (referred to as Amot ACCH domain elution buffer).
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10

Lipid Membrane Composition and Peptide Incorporation

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Lipids including 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and Chol were purchased from Avanti Polar Lipids (Alabaster, AL). 5-doxyl stearic acid (5-SASL) was purchased from Sigma-Aldrich (St. Louis, MO). The 16-residue peptide M2AH of sequence F47FKSIYRFFEHGLKRG62 was synthesized by Genscript. The vendor confirmed peptide purity >90% by HPLC and mass spectroscopy. Fresh peptide stock solution was prepared by dissolving lyophilized powder in 10 mM HEPES pH 7.4. Unless noted otherwise, all ratios and percentages are molar based in this paper.
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