LUVs were prepared by extrusion. Phospholipids composed of bSM/bPC/bPS/cholesterol (2:1:1:1) were dissolved in a mixture of chloroform and methanol, and the solvent was evaporated under a stream of nitrogen gas in a glass test tube. The thin lipid film was further dried overnight under vacuum and hydrated with 0.5 ml of Hepes buffer [10 mM Hepes and 120 mM NaCl (pH 7.2)]. The suspension was vigorously vortexed for 5 min, was subjected to 10 freeze-thaw cycles, and was then extruded 21 times through two stacked polycarbonate filters with a pore size of 100 nm (Avestin). The lipid mixing assay was based on fluorescence resonance energy transfer between NBD-PE and rhodamine-PE. The HIV fusion peptide was added to 50 μM LUVs with a ratio of 1:9 of labeled (1 mol % of NBD-PE and rhodamine-PE each) to unlabeled LUVs in Hepes buffer at room temperature. The fluorescence was recorded under constant stirring in a Fluorolog-3 spectrofluorometer (Jobin-Yvon) with the excitation and emission wavelengths set at 460 and 535 nm, respectively. The value for 0% lipid mixing was the fluorescence intensity of the LUV suspension before the fusion peptide was added, whereas the value for 100% lipid mixing was obtained by adding Triton X-100 [final concentration, 0.5% (v/v)] to the suspension at the end of each experiment.
Polycarbonate filter
Manufactured by Avestin
Sourced in Canada
Polycarbonate filters are a type of laboratory equipment designed to separate and isolate particles or substances from a liquid or gas sample. They are typically made of a thin, porous membrane that allows the passage of the desired components while retaining the unwanted materials. The filters are available in various pore sizes to accommodate different applications and sample requirements.
Automatically generated - may contain errors
Lab products found in correlation
Fluorolog 3 spectrofluorometer, by Horiba (2 mentions)
Lsm 510 meta confocor3, by Zeiss (1 mentions)
Dope cap biotin, by Avanti Polar Lipids (1 mentions)
Filter count, by PerkinElmer (1 mentions)
Tri carb 3110tr scintillation counter, by PerkinElmer (1 mentions)
Thermomixer, by Eppendorf (1 mentions)
11 protocols using polycarbonate filter
1
Liposome Fusion Assay Protocol
2
Preparation and Characterization of LUVs
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Large unilamellar vesicles (LUVs) were prepared by extrusion through polycarbonate filters. In brief, the desired amount of lipids dissolved in chloroform or chloroform/methanol was evaporated under a stream of nitrogen gas in a glass test tube and further dried under high vacuum overnight. The lipid film was hydrated with HEPES buffer (10 mM HEPES, 150 mM NaCl, pH 7.2) and then vortexed. The resulting suspension was subjected to ten cycles of freezing and thawing and thereafter extruded 21 times through two stacked polycarbonate filters with pores of 100 nm in diameter (Avestin, Ottawa, ON). Binding of NBD-labeled recoverin to LUVs was measured as described33 (link). In short, LUVs were added successively to 0.1 μM NBD-recoverin in HEPES buffer. The fluorescence emission spectra in vesicles of different lipid composition were recorded using a Fluorolog-3 spectrofluorometer (Jobin-Yvon, Edison, NJ) with the excitation and emission wavelengths set at 475 nm and 530 nm, respectively.
3
Preparation of Giant Unilamellar Vesicles
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Large unilamellar vesicles (LUVs) were prepared by the extrusion technique. In brief, the desired amounts of lipids dissolved in chloroform or chloroform/methanol were mixed and the solvent was evaporated under a stream of nitrogen gas in a glass test tube. The lipid film was further dried under vacuum overnight and hydrated with 0.5 ml HEPES buffer (10 mM HEPES, 120 mM NaCl, pH 7.2). The resulting suspension was subjected to ten cycles of freezing and thawing using liquid nitrogen and warm water and thereafter extruded 21 times through two stacked polycarbonate filters of 100 nm pore-size (Avestin, Ottawa, ON). Giant unilamellar vesicles (GUVs) were prepared by the electroformation technique51 . In brief, 25 l of a 10 mM lipid solution in organic solvent containing the fluorescent lipid probe Rh-PE (0.1 mol%) was deposited on clean glass slides that were coated with indium tin oxide and then placed in vacuum for 90 min to eliminate residual solvent. The fabrication chamber filled with 300 mM sucrose in H2O was composed by two conducting slides separated by a spacer of 0.5 mm. Electroformation was performed at around 60°C by applying alternating electric current (3 V, 10 Hz) for 120 min. The GUVs were transferred into a 300 mM glucose solution to let them settle by gravity on the microscope slide.
4
Covalent Labeling of Membrane Proteins
5
Measuring D-Aspartate Uptake in Proteoliposomes
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Proteoliposomes (0.1 mg GltPh/ml) were loaded with 200 mM KCl, 20 mM Hepes/KOH (pH 7.4) by three freeze/thaw cycles and extrusion through 400‐nm pore size polycarbonate filters (Avestin). The uptake reaction was performed in an Eppendorf Thermomixer and initiated by the addition of 480 μl uptake buffer (200 mM NaCl, 20 mM Hepes/NaOH pH 7.4, 1 μM valinomycin, 156 nM 3H‐D‐aspartate) to 20 μl proteoliposomes. Uptake buffer and proteoliposomes were pre‐equilibrated at 30°C; after the addition of uptake buffer, the mixture was briefly mixed. At a given time point, the reactions were pipetted on pre‐washed filters (0.22‐μm pore size; GSWP; Millipore) and stopped by immediate suction under vacuum. The filters were washed with 2.5 ml 200 mM LiCl, 20 mM Hepes/NaOH (pH 7.4), mixed with 10 ml Filter‐Count (PerkinElmer), and assayed for radioactivity using a TRI‐CARB 3110 TR scintillation counter (PerkinElmer). Protein was quantified using an amido black assay.
6
Preparation of Giant Unilamellar Vesicles
7
Reconstitution of ZntB in Proteoliposomes
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Reconstitution in proteoliposomes was performed as follows (please see ref. 38 (link) for details): polar lipids of E. coli and egg phosphatidylcholine (in 3:1 (w/w) ratio) were dissolved in chloroform, then dried in a rotary evaporator and subsequently resuspended in buffer containing 50 mM KPi, pH 7.5 to the concentration of 20 mg ml−1. After three freeze-thaw cycles, large unilamellar vesicles (LUVs) were obtained and stored in liquid nitrogen. To prepare proteoliposomes, LUVs were extruded through a 400-nm-diameter polycarbonate filter (Avestin, 11 passages). Obtained liposomes were diluted to 4 mg ml−1 in buffer H (50 mM HEPES, pH 7.5) or buffer I (50 mM HEPES, pH 6.5) and subsequently destabilized beyond Rsat with Triton X-100. Purified ZntB was added to the liposomes at a weight ratio of 1:250 (protein/lipid), followed by detergent removal using Bio-beads (50 mg ml−1, four times after 0.5 h, 1 h, 2 h and overnight incubation). Afterwards, proteoliposomes were collected by centrifugation (25 min, 285,775×g, 4 °C) and resuspended in buffer H or buffer I to a lipid concentration of 10 mg ml−1. Finally, after three freeze-thaw cycles, obtained proteoliposomes were stored in liquid nitrogen until subsequent experiments.
8
Reconstitution of Proteins in Proteoliposomes
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Reconstitution in proteoliposomes was performed as described previously18 : polar lipids of E. coli and egg phosphatidylcholine (in 3:1 (w/w) ratio) were dissolved in chloroform, then dried in a rotary evaporator and subsequently resuspended in buffer containing 50 mM KPi, pH 7.5 to the concentration of 20 mg ml−1. After three freeze-thaw cycles, large unilamellar vesicles (LUVs) were obtained and stored in liquid nitrogen. To prepare proteoliposomes, LUVs were extruded through a 400-nm-diameter polycarbonate filter (Avestin, 11 passages). Obtained liposomes were diluted to 4 mg ml−1 in buffer H (50 mM HEPES, pH 7.5) or buffer I (50 mM HEPES, pH 6.5) and subsequently destabilized beyond Rsat with Triton X-100. The target purified protein was added to the liposomes at a weight ratio of 1:250 (protein/lipid), followed by detergent removal using Bio-beads (50 mg ml−1, four times after 0.5 h, 1 h, 2 h and overnight incubation). Afterwards, proteoliposomes were collected by centrifugation (25 min, 285,775 g, 4 °C) and resuspended in buffer H or buffer I to a lipid concentration of 10 mg ml−1. Finally, after three freeze-thaw cycles, obtained proteoliposomes were stored in liquid nitrogen until subsequent experiments.
9
Lipid Vesicle Preparation and Peptide Binding Assay
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POPG, dimyristoylphosphatidylcholine, or dipalmitoylphosphatidylcholine (Avanti Polar Lipids), were prepared by extrusion as reported previously (66 (link)). The phospholipids solution was transferred to a glass vial, and the solvent was evaporated under a stream of nitrogen. The vial was placed in the lyophilizer under vacuum overnight. The lipid film was resuspended by adding PBS buffer to obtain a multilamellar vesicle suspension with a POPG concentration of 1 mg/ml. The lipid solution was subjected to a water bath sonication to disrupt the multilamellar vesicles. LUVs were then prepared by extrusion, applying 21 passes through a polycarbonate filter with a 100-nm average pore diameter (Avestin). Samples for membrane-binding studies were prepared by adding 5 molar eq of lipid to the Nt17 peptide solution (60 μm final concentration) and incubating the peptide/LUV samples at room temperature for 1 h before CD measurements.
10
Reconstitution of Membrane Proteins
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An aliquot of 20 mg/mL E. coli polar lipid extract in 50 mM potassium phosphate buffer (KPi) pH 7.0 was extruded by 11 passages through a 400 nm diameter polycarbonate filter (Avestin). After diluting the lipid mixture to 4 mg/mL using the same buffer, it was destabilised with 10% Triton-X100 following the absorption at 540 nm (Jasco). The titration was stopped once the absorption signal decreased to about 60% of the maximum value reached. The two protein variants were then separately added to the solutions in two different protein:lipid ratios: 1:800 (w/w) for the radioactive assays and 1:75 (w/w) for SSM, and incubated for 30 min at 20 °C on a rocking platform. In order to remove detergent, Bio-beads were increasingly added to the mixture and incubated at 4 °C in this order: 25 mg/mL for 30 min, 15 mg/mL for 1 h, 19 mg/mL overnight and 4 mg/mL for 2 h. After removing Bio-beads, the proteoliposomes were centrifuged at 298,906 × g at 4 °C for 20 min, resuspended to 20 mg/mL in internal lumen buffer (different for radioactivity measurements and SSM – see the following paragraphs). Internal buffer was exchanged by three cycles of freeze-thawing. Finally, the aliquots were stored in liquid nitrogen until further use.
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