Polycarbonate filter

Manufactured by Avanti Polar Lipids

Sourced in United States

Polycarbonate filters are a type of lab equipment designed to separate particles or molecules from a liquid or gas sample. They are made of polycarbonate, a durable and transparent material. Polycarbonate filters are used to filter and purify samples in various applications, such as cell culture, water treatment, and sample preparation.

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Lab products found in correlation

Mini extruder, by Avanti Polar Lipids (6 mentions) Microcentrifuge, by Eppendorf (2 mentions) Dogs nta, by Avanti Polar Lipids (1 mentions) 1 1 dioctadecyl 3 3 3 3 tetramethylindocarbocyanine perchlorate dii, by Thermo Fisher Scientific (1 mentions) 1 1 dioctadecyl 3 3 3 3 tetramethylindodicarbocyanine perchlorate did, by Thermo Fisher Scientific (1 mentions) Pi 3 p, by Avanti Polar Lipids (1 mentions) D 78224, by Elma (1 mentions) Ergosterol, by Merck Group (1 mentions) Triton x 100, by Anatrace (1 mentions) 70.1 ti rotor, by Beckman Coulter (1 mentions) Infinite m200, by Tecan (1 mentions) Lecithin, by Avanti Polar Lipids (1 mentions) Dipalmitoyl phosphatidylinositol 3 phosphate pi3p, by Echelon Biosciences (1 mentions) L α phosphatidylcholine, by Merck Group (1 mentions) Speedvac, by Thermo Fisher Scientific (1 mentions) Atto488 dope, by ATTO-TEC (1 mentions)

34 protocols using polycarbonate filter

Liposome Preparation for Membrane Tethering

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All of the non-fluorescent lipids for liposome preparation, including POPC (1-palmitoyl-2-oleoyl-phosphatidylcholine), POPE
(1-palmitoyl-2-oleoyl-phosphatidylethanolamine), liver PI (phosphatidylinositol), POPS (1-palmitoyl-2-oleoylphosphatidylserine), cholesterol, and DOGS-NTA (1,2dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl) iminodiacetic acid]-succinyl}), were from Avanti Polar Lipids. The two fluorescence-labeled lipids, Rh-PE (rhodamine-PE) and FL-PE (fluorescein-PE), were from Molecular Probes. Lipid mixes used were prepared in chloroform with the lipid compositions of 41% (mol/mol) POPC, 17% POPE, 10% liver PI, 5% POPS, 20% cholesterol, 6% DOGS-NTA, and 1% Rh-PE or FL-PE, dried up by evaporating chloroform with a stream of nitrogen gas, and subsequently resuspended in RB150 containing 5 mM MgCl 2 and 1 mM DTT (final 8 mM lipids) by vortexing vigorously and incubating with agitation (37°C, 1 h). After freeze-thawing in liquid nitrogen and a water bath at 30°C, lipid suspensions were extruded 25 times through polycarbonate filters (pore diameters, 200 nm; Avanti Polar Lipids) in a mini-extruder (Avanti Polar Lipids) preheated at 40°C. The liposome solutions prepared were stored at 4°C Arf6-mediated membrane tethering 3 and used within a week for liposomes turbidity assays and fluorescence microscopy.

Fujibayashi K., & Mima J. (2020). The small GTPase Arf6 functions as a membrane tether in a chemically-defined reconstitution system.

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Preparation of FRET-Labeled Liposomes

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Lipids in chloroform were dried under a stream of N₂ gas followed by further drying in the vacuum for 2 hours. Moles percent of lipids used for the acceptor and donor liposomes in FRET-based lipid transfer experiments were as indicated in Supplementary Table 3.
The dried lipid films were hydrated with buffer (25 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.5 mM TCEP). Liposomes were then formed by ten freeze-thaw cycles (liquid N₂ and 37°C water bath) followed by extrusion through polycarbonate filters with a pore size of 50 nm (Avanti Polar Lipids).

Saheki Y., Bian X., Schauder C.M., Sawaki Y., Surma M.A., Klose C., Pincet F., Reinisch K.M, & De Camilli P. (2016). Control of plasma membrane lipid homeostasis by the extended synaptotagmins. Nature cell biology, 18(5), 504-515.

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Preparation of DNA-free Liposomes

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DNA-free donor liposomes were composed as follows: 87:1.5:1.5:10 mole percent of DOPC: NBD-PE: Rhod-PE: DGS-NTA(Ni) or 87:1.5:1.5:10 mole percent of DOPC: NBD-PS: Rhod-PE: DGS-NTA(Ni). DNA-free acceptor liposomes were composed as follows: 85:10:5 mole percent DOPC: POPS: PI(4,5)P₂. Liposome preparation was performed as previously described^{24 (link)}. Briefly, lipid mixtures were dried onto a film. Lipid films were then hydrated with buffer A. DNA-free liposomes were generated by ten freeze-thaw cycles in liquid N₂ and 37 °C water bath. LUVs were formed by extrusion through polycarbonate filters with a pore size of 50, 100 or 400 nm (Avanti Polar Lipids). SUVs were formed by sonication for 20 min using a probe tip sonicator (Virtis Virsonic).

Bian X., Zhang Z., Xiong Q., Camilli P.D, & Lin C. (2019). A programmable DNA-origami platform for studying lipid transfer between bilayers. Nature chemical biology, 15(8), 830-837.

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Characterization of Pneumolysin-Membrane Interactions

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Pneumolysin was expressed and purified as previously described [56 (link)]. Pneumolysin was incubated with single unilamellar vesicles (SUVs) containing egg phosphatidylcholine : cholesterol : dicetyl phosphate in molecular ratios of 10 : 10 : 1 at a cholesterol to protein ratio of 100 : 1. Note however that, as shown by the tomograms determined below (see the electronic supplementary material, Movies S1 and S2), some of the SUVs were double layered. SUVs were prepared as follows: a fine lipid film was formed under a stream of argon, dried in a desiccator, solubilized with sterile phosphate buffered saline, and subjected to 10 iterative freeze–thaw cycles followed by repetitive extrusion (at least 11 passes) at 37°C through 100 nm polycarbonate filters (Avanti Polar lipids). Protein/liposome incubations were performed at 37°C for 1 min or 5 s (directly on the EM grid in a humidity chamber) as reported previously [28 (link)]. Longer timeframes result in sample aggregation [28 (link),31 (link)]. Prior to application of the proteoliposomal suspension and vitrification in liquid ethane by plunge freezing, lacey carbon or C-Flat copper grids (Plano, Wetzlar, Germany) were loaded with a suspension containing 10 nm colloidal gold clusters.

Sonnen A.F., Plitzko J.M, & Gilbert R.J. (2014). Incomplete pneumolysin oligomers form membrane pores. Open Biology, 4(4), 140044.

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Preparation of Endosome-Mimicking Liposomes

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LUVs were prepared with a lipid composition either of DOPC or mimicking the membranes of early and late endosomes. For early endosome-mimicking LUVs, the lipid content was DOPC:DOPE:SM:chol 30:10:25:35, while late endosome-mimicking LUVs were composed of LBPA:DOPE:DOPC 70:5:25 (molar ratio) to mimic the cholesterol sequestration and enrichment of late endosomal lipid LBPA (49) . Lipid solutions in chloroform of the different phospholipid species were mixed to the desired molar ratios in a glass vial, and the organic solvent was evaporated by 12 h of vacuum pumping. For labeled LUVs, the lipids were stained at a 2 % mol/mol fraction of DiI and DiD in chloroform before evaporation. The lipid film was then hydrated with PBS -/-at 50 °C to reach the desired concentration and gently vortexed. The resulting MLV suspension was then sonicated for 10 min to disperse larger aggregates and the liposomal suspension was extruded 21 times through polycarbonate filters (100 nm pore size, Avanti Polar Lipids) using a mini-extruder (Avanti Polar Lipids). Size and concentration were verified using NTA and the liposomal suspension was used within 2 weeks from extrusion.

Morandi M.I., Busko P., Ozer-Partuk E., Khan S., Zarfati G., Elbaz‐Alon Y., Karam P.A., Shogan T.N., Ginini L., Gil Z., Regev‐Rudzki N., & Avinoam O. (2022). Extracellular vesicle fusion visualized by cryo-EM.

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Preparation of Liposomes Mimicking Red Blood Cell Membrane

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LUVs were prepared with a lipid composition of DOPC:DOPE:DOPS:SM:chol of 20:5:15:25:35 (molar ratio), resembling the RBC plasma membrane composition as previously described^{33 (link)}. Lipid solution in chloroform of the different phospholipid species were mixed to the desired molar ratios in a glass vial, and organic solvent was evaporated by 12 h of vacuum pumping. For labeled LUVs, the lipids were stained with 2% mol of DiI and DiD in chloroform before evaporation. The lipid film was then hydrated with PBS (Ca²⁺-/Mg²⁺-at 40 °C to reach the desired concentration and gently vortexed. The resulting MLV suspension was then sonicated for 10 min to disperse larger aggregates and the liposomal suspension was extruded 21 times through polycarbonate filters (100 nm pore size, Avanti Polar Lipids) using a mini-extruder (Avanti Polar Lipids). Size and concentration were verified using NTA and the liposomal suspension was used within 2 weeks from the extrusion. DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) and DiD (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindodicarbocyanine 4-Chlorobenzenesulfonate Salt) membrane dyes were purchased from Thermo Fisher as a powder and dissolved in chloroform at 1 mM final concentration. All chemicals had high purity and were used without further purification.

Dekel E., Yaffe D., Rosenhek-Goldian I., Ben-Nissan G., Ofir-Birin Y., Morandi M.I., Ziv T., Sisquella X., Pimentel M.A., Nebl T., Kapp E., Ohana Daniel Y., Karam P.A., Alfandari D., Rotkopf R., Malihi S., Temin T.B., Mullick D., Revach O.Y., Rudik A., Gov N.S., Azuri I., Porat Z., Bergamaschi G., Sorkin R., Wuite G.J., Avinoam O., Carvalho T.G., Cohen S.R., Sharon M, & Regev-Rudzki N. (2021). 20S proteasomes secreted by the malaria parasite promote its growth. Nature Communications, 12, 1172.

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IFITM3 Reconstitution into Liposomes

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Lipids (99.0 mol % POPC, 0.5 mol % cholesterol, 0.5 mol % Liss-Rho-PE) were mixed in a glass tube and dried down to a film under a gentle stream of Argon, followed by further drying under a vacuum for 30 min. Next, the lipid film was hydrated and resuspended with HND buffer to a final lipid concentration of 10 mM. Large unilamellar vesicles (LUVs) were formed from the lipid suspension by ten freeze-thaw cycles using liquid nitrogen and room temperature water bath. Uniform-sized LUVs were formed by extruding through polycarbonate filters with 100-nm pore size (Avanti Polar Lipids, Alabaster, AL) 11 times.
To reconstitute IFITM3, preformed liposomes and purified IFITM3 (molar protein to lipid ratio, 1:500) were mixed with 0.1% Triton X-100 at an effective detergent to lipid ratio of ~1 and incubated for 1h at 4 °C. Triton X-100 was then removed by adding BioBeads SM-2 absorbent beads (BioRad) at a Bio-Beads/Triton X-100 ratio of 10 (wt/wt) in five portions during the course of hour, and incubating overnight after the final addition of beads. Insoluble protein aggregates were pelleted by centrifugation of samples in an Eppendorf microcentrifuge (10 min, 16,000×g).

Guo X., Steinkühler J., Marin M., Li X., Zhang Y., Dimova R., & Melikyan G.B. (2020). Interferon-Induced Transmembrane Protein 3 Blocks Fusion of Diverse Enveloped Viruses by Locally Altering Mechanical Properties of Cell Membranes.

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Reconstitution of Protein-Lipid Complexes

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The lipids used in this study were phosphatidylcholine (POPC), phosphatidylethanolamine (POPE), biotinylated phosphatidylethanolamine (biotin-PE) and PI3P (Avanti Polar Lipids). Also, 2–4 mol% 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) or 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) (Molecular Probes) were used as lipid dyes for the v- and t-proteoliposomes, respectively. The lipid mixtures POPE:POPC:DiI (molar ratio 20:78–76:2–4) or POPE:POPC:PI3P:DiI (molar ratio 20:76–74:2:2–4) were solubilized in chloroform and then dried to form a lipid film on the wall of a glass tube. The same procedures were applied to prepare another aliquot of lipid solution containing the same ratio of PE, PC, DiD, supplemented with 0.1 mol% biotinylated lipid (or with 2% PI3P). The dried lipid film was resuspended in proteoliposome buffer (20 mM HEPES, 100 mM KCl, pH 7.4). After five freeze–thaw cycles, unilamellar proteoliposomes were extruded through polycarbonate filters (50 nm pore size, Avanti Polar Lipids) at least 39 times.

Diao J., Liu R., Rong Y., Zhao M., Zhang J., Lai Y., Zhou Q., Wilz L.M., Li J., Vivona S., Pfuetzner R.A., Brunger A.T, & Zhong Q. (2015). ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes. Nature, 520(7548), 563-566.

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Preparation of Ni-NTA Liposomes

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Phospholipids (79.7% POPC + 10% POPS + 10% DGS-NTA-Ni + 0.3% Rhod-PE) were dried under a stream of N₂, desiccated for 3 h and suspended in 1x Kinase buffer. Large unilamellar liposomes were prepared by extrusion through a pair of polycarbonate filters with a pore size of 200 nm, according to the manufacturer instructions (Avanti Polar Lipids). For liposomes used in Supplementary Fig. 1c, the changes in PS content were compensated by opposite changes in PC content.

Hui E, & Vale R.D. (2014). In vitro membrane reconstitution of the T cell receptor proximal signaling network. Nature structural & molecular biology, 21(2), 133-142.

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Quantification of VSTx1 Binding to Lipid Vesicles

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Large unilamellar vesicles (LUVs) were prepared by drying lipids (POPG and/or POPC, 25 mg/mL) under nitrogen gas, drying the lipid pellet under vacuum, and resuspending the lipids by vortexing for 1 h in an aqueous buffer containing 10 mM HEPES pH 7.4, and 150 mM NaCl. Vesicles were extruded 20 times using 0.1 μm polycarbonate filters (Avanti Polar Lipids, Inc.). Aqueous solutions of the toxin (10 μM) were incubated with varying amounts of vesicles for 1 h at room temperature, then the vesicles were centrifuged for 30 min at 100,000 g before the toxin concentration in the aqueous phase was determined. Aqueous concentrations of VSTx1 were quantified by RP-HPLC using a monolithic C₁₈ column (4.6 × 100 mm). The toxin was eluted with a linear gradient of 10–50% solvent B (0.046% TFA in acetonitrile) in solvent A (0.05% TFA in water) over 10 min at a flow rate of 3 ml/min. The partition coefficients (K_p) for the binding of VSTx1 to liposomes were calculated by fitting to the partition equilibrium equation:

Lau C.H., King G.F, & Mobli M. (2016). Molecular basis of the interaction between gating modifier spider toxins and the voltage sensor of voltage-gated ion channels. Scientific Reports, 6, 34333.

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