A mixture of porcine brain polar lipid extract dissolved in chloroform and CHS (Avanti Polar Lipids, USA) at a ratio of 4:1 (w/w) was dried under a gentle stream of nitrogen. The lipid thin film was placed under a desiccator overnight to completely remove residual chloroform and then hydrated in reconstitution buffer containing 20 mM HEPES pH 7.5 and 100 mM KCl. After sonication, the lipid mixture was extruded 15 times through a 200 nm polycarbonate membrane filter (Avanti Polar Lipids) to form unilamellar vesicles and then destabilized with 0.056% (w/v) Cymal-6. The purified protein and lipid suspension were mixed in a ratio of 1:25 (w/w). After incubation at 4°C for 2 h, the detergent was removed with Bio-Beads SM-2 resin (Bio-Rad, USA). Proteoliposomes were collected by centrifugation at 200,000 × g for 1 h and resuspended in reconstitution buffer for transport assays. Protein-free liposomes were prepared in parallel.
200 nm polycarbonate membrane filter
Manufactured by Avanti Polar Lipids
Sourced in United States
The 200 nm polycarbonate membrane filter is a laboratory filtration device designed to separate particles or molecules based on size. It has a pore size of 200 nanometers, allowing it to effectively filter out smaller components from a solution while retaining larger ones.
Automatically generated - may contain errors
3 protocols using 200 nm polycarbonate membrane filter
1
Proteoliposome Formation for Transport Assays
2
Preparation of Carboxylate Polystyrene Particles and Liposomes
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Carboxylate polystyrene
particles (2% solids) were diluted in a 0.1 mM LiCl solution at a
particle concentration of 0.002% (v/v) for the 1 μm particles
and 0.02% (v/v) for particles of other diameters. Liposomes were prepared
using standard thin film hydration73 (link) and
subsequent extrusion techniques.74 (link) Briefly,
lipids and lipophilic dye (DiOc18) were dissolved in chloroform and mixed at appropriate ratios.
The chloroform was evaporated by using a constant flow of N2. The dried lipid film was desiccated for a minimum of 3 h before
hydration and dilution using a 0.01 mM LiCl + 5 μM EDTA + 5
μM HEPES solution, pH = 8.15. The liposome suspension was then
vortexed at 1200 rpm for 60s and extruded 21 times through a 200 nm
polycarbonate membrane filter (Avanti Polar Lipids, US) to maintain
an average polydispersity index (PDI) of 0.1.
particles (2% solids) were diluted in a 0.1 mM LiCl solution at a
particle concentration of 0.002% (v/v) for the 1 μm particles
and 0.02% (v/v) for particles of other diameters. Liposomes were prepared
using standard thin film hydration73 (link) and
subsequent extrusion techniques.74 (link) Briefly,
lipids and lipophilic dye (DiOc18) were dissolved in chloroform and mixed at appropriate ratios.
The chloroform was evaporated by using a constant flow of N2. The dried lipid film was desiccated for a minimum of 3 h before
hydration and dilution using a 0.01 mM LiCl + 5 μM EDTA + 5
μM HEPES solution, pH = 8.15. The liposome suspension was then
vortexed at 1200 rpm for 60s and extruded 21 times through a 200 nm
polycarbonate membrane filter (Avanti Polar Lipids, US) to maintain
an average polydispersity index (PDI) of 0.1.
3
Reconstitution of Membrane Proteins in Liposomes
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Liposomes were prepared using a 1:1 (w/w) ratio of Egg PC and E. coli polar lipid extract (Avanti Polar Lipids). Lipids solubilized in chloroform were dried under nitrogen gas and resuspended in buffer containing 20 mM HEPES-NaOH pH 7.0 and 200 mM NaCl. After five freeze-thaw cycles, the liposome suspension was passed through a Mini-Extruder equipped with a 200 nm polycarbonate membrane filter (Avanti Polar Lipids) to produce unilamellar liposomes. Liposomes were destabilized with 5 mM n-Decyl-β-D-Maltopyranoside (DM; Anatrace) at a 1:1 (mol/mol) ratio for 3 h at 22 °C. The purified protein was added to the mixture at a 1:50 (w/w) protein to lipid ratio and incubated for 2 h at 4 °C with gentle shaking. Detergent was removed by adding Bio-Beads (200 mg/mL) for 2 h at 4 °C followed by overnight incubation with fresh Bio-Beads. The supernatant was further treated with fresh Bio-Beads for 2 h, and proteoliposomes were separated by Histodenze density gradient centrifugation (35%: 25%: 0%; Sigma-Aldrich) at 259,000 g for 1 h at 4 °C51 (link). The resulting proteoliposomes were diluted in buffer comprising 20 mM HEPES-NaOH pH 7.0 and 200 mM NaCl to a final phospholipid concentration of 0.5 mM. Liposomal lipid concentrations were determined by colorimetric phosphate assay52 (link).
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