POPC proteoliposomes were prepared as reported [59 (link)] from detergent solubilized and BODIPY-C3-M labeled LeuT using a protein:lipid (w:w) ratio of 1:100. In brief POPC lipid (Avanti Polar lipids Inc.) was dried under a gentle stream of nitrogen to remove the organic solvent chloroform, remaining chloroform traces were removed overnight in a rotavapor. The lipid film was dissolved in 200 mM KCl or NaCl buffer and 20 mM HEPES at pH 7.5 to a final concentration of 20 mg/ml. The lipid suspension was then sonicated for 45 minutes (using three 15-min cycles), flash frozen and slowly thawed to room temperature. Liposomes were extruded 11 times with a mini extruder (Avanti lipids) over a filter of pore size of 400 nm. Liposomes were destabilized with Triton X-100 and detergent solubilized LeuT added at a 1:100 protein:lipid ratio and incubated at room temperature for 30 min. Detergent was removed using biobeads (Biorad) followed by ultracentrifugation at 120000g for 90 min. The proteoliposomes were re-suspended to a final concentration of 100 mg/ml and stored at -80°C until use.
Popc lipid
Manufactured by Avanti Polar Lipids
Sourced in France, United States, United Kingdom
POPC lipids are a type of phospholipid commonly used in liposome preparation and membrane research. They are composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and serve as a standard model for investigating the properties and behavior of lipid membranes.
Automatically generated - may contain errors
Lab products found in correlation
Bio beads, by Bio-Rad (1 mentions)
Ifs 55 infrared spectrophotometer, by Bruker (1 mentions)
Bio beads sm 2, by Bio-Rad (1 mentions)
α naphthoflavone, by Cayman Chemical (1 mentions)
Midazolam, by Cayman Chemical (1 mentions)
Gefitinib, by Cayman Chemical (1 mentions)
0.1 μm polycarbonate membrane, by Avanti Polar Lipids (1 mentions)
Oregon green 488, by Thermo Fisher Scientific (1 mentions)
Oregon green 488 dhpe, by Thermo Fisher Scientific (1 mentions)
7 protocols using popc lipid
1
POPC Proteoliposome Preparation
2
Carboxyfluorescein-Encapsulated Liposome Preparation
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The nanovesicles (liposomes) were prepared by
mixing POPC lipid (Avanti Polar Lipids), synthesized lipopeptide LP,
POPE-SS-PEG5000, and cholesteryl hemisuccinate in molar
proportions of 60:30:5:5, respectively. All the lipids were dissolved
in chloroform. The chloroform was removed using a rotary evaporator
to form a thin lipid film in a round-bottom flask. The film was further
vacuum-dried overnight inside a desiccator. The film was then hydrated
at 60 °C for 2 h with 100 mM carboxyfluorescein solution prepared
in HEPES buffer (pH 7.4). The formed vesicles were subjected to ultrasonication
for 45 min using an Aquasonic bath sonicator (model 250D, power level
9). The resulting vesicles were then extruded through 0.8 μm
and, subsequently, 0.2 μm filters to obtain vesicles with a
uniform size. To remove the unencapsulated dye, the vesicles were
passed through a Sephadex G50-size exclusion column, and an orange
band of carboxyfluorescein-encapsulated nanovesicles was collected.
These vesicles were used for the release and imaging experiments.
Since a large excess of carboxyfluorescein was used, we did not estimate
the percentage of the dye encapsulated.
mixing POPC lipid (Avanti Polar Lipids), synthesized lipopeptide LP,
POPE-SS-PEG5000, and cholesteryl hemisuccinate in molar
proportions of 60:30:5:5, respectively. All the lipids were dissolved
in chloroform. The chloroform was removed using a rotary evaporator
to form a thin lipid film in a round-bottom flask. The film was further
vacuum-dried overnight inside a desiccator. The film was then hydrated
at 60 °C for 2 h with 100 mM carboxyfluorescein solution prepared
in HEPES buffer (pH 7.4). The formed vesicles were subjected to ultrasonication
for 45 min using an Aquasonic bath sonicator (model 250D, power level
9). The resulting vesicles were then extruded through 0.8 μm
and, subsequently, 0.2 μm filters to obtain vesicles with a
uniform size. To remove the unencapsulated dye, the vesicles were
passed through a Sephadex G50-size exclusion column, and an orange
band of carboxyfluorescein-encapsulated nanovesicles was collected.
These vesicles were used for the release and imaging experiments.
Since a large excess of carboxyfluorescein was used, we did not estimate
the percentage of the dye encapsulated.
3
ATR-FTIR Spectroscopic Analysis of Peptide-Lipid Interactions
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ATR-FTIR spectra were recorded on a Bruker IFS
55 infrared spectrophotometer (Ettlingen, Germany) equipped with a
liquid-nitrogen-cooled MCT detector. The internal reflection element
(IRE) was a germanium (Ge) ATR plate (50 × 20 × 2 mm3) with an aperture angle of 45°, yielding 25 internal
reflections. A total of 128 scans were accumulated for each spectrum
to improve the signal-to-noise ratio. Spectra were recorded at a nominal
resolution of 2 cm–1. The spectrophotometer was
continuously purged with dried air. All of the measurements were carried
out at 24 °C.
APP_TM4K peptides were dissolved in HFIP
(Sigma-Aldrich Chimie S.a.r.l., Lyon, France) to a final concentration
of 2 mg/mL. This solution was mixed with POPC lipids (Avanti Polar
Lipids, Alabaster, AL) dissolved in chloroform (Sigma-Aldrich Chimie
S.a.r.l., Lyon, France) to obtain a 1:50 peptide-to-lipid ratio. The
solvents were evaporated, and the sample was spread on the surface
of the Ge plate to form a thin film of oriented multilayers.
55 infrared spectrophotometer (Ettlingen, Germany) equipped with a
liquid-nitrogen-cooled MCT detector. The internal reflection element
(IRE) was a germanium (Ge) ATR plate (50 × 20 × 2 mm3) with an aperture angle of 45°, yielding 25 internal
reflections. A total of 128 scans were accumulated for each spectrum
to improve the signal-to-noise ratio. Spectra were recorded at a nominal
resolution of 2 cm–1. The spectrophotometer was
continuously purged with dried air. All of the measurements were carried
out at 24 °C.
APP_TM4K peptides were dissolved in HFIP
(Sigma-Aldrich Chimie S.a.r.l., Lyon, France) to a final concentration
of 2 mg/mL. This solution was mixed with POPC lipids (Avanti Polar
Lipids, Alabaster, AL) dissolved in chloroform (Sigma-Aldrich Chimie
S.a.r.l., Lyon, France) to obtain a 1:50 peptide-to-lipid ratio. The
solvents were evaporated, and the sample was spread on the surface
of the Ge plate to form a thin film of oriented multilayers.
4
Nanodisc Preparation for Cryo-EM
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The preparation of nanodiscs was performed by mixing 8C-8A(IL125) purified in the presence of DDM with POPC lipids (Avanti Polar Lipids, Inc) and MSP1E3D1 at a final molar ratio of 1:2.5:250 (8C-8A(IL125):MSP1E3D1:POPC). The mixture was incubated with Biobeads SM2 (Bio-Rad) overnight. After removing the biobeads by centrifugation, the protein sample was concentrated and purified by SEC using a Superose 6 10/300 Increase column equilibrated with 150 mM NaCl and 50 mM Tris-HCl, pH 8.0. Peak fractions corresponding to 8C-8A(IL125)-MSP1E3D1 nanodiscs were concentrated to 2.0 mg/ml and used for cryo-EM grid preparation immediately.
5
Quantification of Steroid and Drug Compounds
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Androstenedione (9001311), corticosterone (16063), medroxyprogesterone (24908), progesterone (15876), testosterone (15645), gefitinib (13166), schisandrin A (19849), α-naphthoflavone (16924), and midazolam (16193) were purchased from Cayman Chemical Company. POPC lipids were purchased from Avanti Polar Lipids. All other chemicals were purchased from Sigma-Aldrich. Structures are shown in the Scheme 1 .
6
POPC Liposome Preparation Protocol
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Powdered 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phospho-choline (POPC) lipids (Avanti Polar Lipids, Alabaster, AL, USA) were resuspended in 50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM CaCl2 to a concentration of 1.3 mM. Resuspended POPC was extruded through an Avanti Mini-Extruder to produce unilamellar liposomes. Briefly, 1 mL of resuspended lipids were solubilized by 15 cycles of rapid freeze–thaws in liquid-nitrogen and a 45 °C water bath. Solubilized lipids were then extruded through a 0.1 μm polycarbonate membrane (Avanti Polar Lipids) 29 times using the Avanti Mini-Extruded apparatus. Extruded liposomes were stored at 4 °C for up to one week.
7
Visualizing Amyloid-beta Binding to Supported Lipid Bilayers
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The SLB consisted of 1 palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids from Avanti Polar Lipids with 0.01 wt% of the fluorescently labelled lipid Oregon Green® 488 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (Oregon Green® 488 DHPE; Invitrogen, UK) to visualize the SLB in order to make sure there were no defects. After forming the SLB, Aβ labelled with HiLyte 647 was added to the dish. This was done at different concentrations (higher concentration leads to more binding), all in L15 media (from 2.5 nM to 250 nM). The binding of the Aβ was imaged with TIRF microscopy (with a HeNe laser operating at 633 nM) either with Aβ in the solution (at 1 s between frames) or after rinsing the solution to remove unbound Aβ (stream acquisition with 100 ms exposure time to measure single-step photobleaching).
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