100 nm polycarbonate membrane

Manufactured by Avanti Polar Lipids

Sourced in United States

The 100 nm polycarbonate membrane is a lab equipment product used for filtration and separation purposes. It is a thin, porous membrane made of polycarbonate material with a nominal pore size of 100 nanometers. The primary function of this membrane is to facilitate the separation and isolation of particles, molecules, or other substances based on their size.

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Lab products found in correlation

Zetasizer nano zs instrument, by Malvern Panalytical (1 mentions) Ht7700, by Hitachi (1 mentions) Zetasizer nano z, by Malvern Panalytical (1 mentions) Pd 10 desalting column, by GE Healthcare (1 mentions) Sulforhodamine b, by Merck Group (1 mentions) Cary eclipse fluorescence spectrophotometer, by Agilent Technologies (1 mentions) Triton x 100, by Merck Group (1 mentions) Dopc 850375c, by Avanti Polar Lipids (1 mentions) Dopg 840475c, by Avanti Polar Lipids (1 mentions) Folch lipids, by Merck Group (1 mentions) Lipid stocks, by Avanti Polar Lipids (1 mentions) Brain sm, by Avanti Polar Lipids (1 mentions)

Close spelling variants of this product

Polycarbonate membranes

9 protocols using 100 nm polycarbonate membrane

Extrusion-Based Liposome Preparation

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Unilamellar
liposomes were prepared by the extrusion method. Lipids in chloroform
were mixed in a glass tube and dried as thin films under a stream
of nitrogen gas. 1–2 mol % of 5-SASL was added to the lipid
mixture. To remove residual organic solvent, the lipid films were
further dried using a vacuum pump for ∼16 h. The lipids were
resuspended in a HEPES buffer (10 mM HEPES, pH 7.4) by vortexing for
1–2 min and then subjected to ≥6 freeze-and-thaw cycles.
The lipid suspension was then extruded ≥30 times through a
mini extruder with a 100 nm polycarbonate membrane (Avanti Polar Lipids).

Pan J., Dalzini A., Khadka N.K., Aryal C.M, & Song L. (2018). Lipid Extraction by α-Synuclein Generates Semi-Transmembrane Defects and Lipoprotein Nanoparticles. ACS Omega, 3(8), 9586-9597.

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Preparation and Characterization of Liposomes

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Liposomes were prepared by classical thin film hydration followed by extrusion (22 (link),38 (link)). Briefly, DOPC, DPPC, and DHPE-Texas Red with a defined molar ratio were dissolved in ∼1.5 mL chloroform in a flask. The solution was then evaporated via vacuum rotary evaporation to form a thin film at the bottom of the flask, followed by vacuum drying overnight to completely remove chloroform. The thin film of phospholipids was then hydrated with buffer (150 mM NaCl in phosphate buffer saline [PBS]) to achieve a final phospholipid concentration of 3 mg mL⁻¹. The phospholipid solution was subjected to 10 freeze–thaw cycles and extruded through a 100-nm polycarbonate membrane (Avanti Polar Lipids) to prepare liposomes. The dynamic light scattering characterization of liposomes were performed on a Malvern Zetasizer Nano ZS instrument. For TEM imaging, 10 μL of liposomes was dropped on a TEM grid for 10 s and negatively stained with 10 μL of 2% uranyl acetate for 10 s. After washing with ultrapure water and drying, the sample was observed by TEM (HT-7700, Hitachi). The prepared liposomes could be stored at 4 °C for further use (preferably use within a week).

Niu Q., Gao J., Zhao K., Chen X., Lin X., Huang C., An Y., Xiao X., Wu Q., Cui L., Zhang P., Wu L, & Yang C. (2022). Fluid nanoporous microinterface enables multiscale-enhanced affinity interaction for tumor-derived extracellular vesicle detection. Proceedings of the National Academy of Sciences of the United States of America, 119(44), e2213236119.

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Lipid Extraction and Vesicle Formation

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Lipids used for experiments were extracted from either E. coli or P. pastoris using the Bligh and Dyer method [36 (link)]. The lipids were then dried under a stream of nitrogen gas and polar lipids were extracted using a cold acetone precipitation [37 (link)]. The E. coli or P. pastoris polar lipids were solubilized by repeated heating and cooling, between 42

°

C and room temperature, in either 10 mM succinate pH 4, 10 mM acetate pH 5, 10 mM MES pH 6, or 10 mM phosphate pH 7. The solution was then run through a mini extruder equipped with a 100 nm polycarbonate membrane (Avanti Polar Lipids, Alabaster, AL, USA) to create large unilamellar vesicles (LUV) of approximately 100 nm in diameter. Vesicle size was monitored over time at the different pH with or without 50

μ

M D1 PSI using dynamic light scattering on a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK).

Sprague-Piercy M.A., Bierma J.C., Crosby M.G., Carpenter B.P., Takahashi G.R., Paulino J., Hung I., Zhang R., Kelly J.E., Kozlyuk N., Chen X., Butts C.T, & Martin R.W. (2020). The Droserasin 1 PSI: A Membrane-Interacting Antimicrobial Peptide from the Carnivorous Plant Drosera capensis. Biomolecules, 10(7), 1069.

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Liposome-Mediated BHA Dequenching Assay

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Liposomes were prepared by mixing DOPC (dioleoyl-phosphatidylcholine) (Avanti Polar Lipids) and bovine brain Calbiochem® disialoganglioside GD_1a (Millipore) in a 100:5 molar ratio, and dried under nitrogen gas. The lipid films were re-suspended in liposome buffer (HBS with 10 mM Na-Citrate, and 25 mM sulforhodamine-B (Sigma-Aldrich)), extruded through a 100 nm polycarbonate membrane (Avanti Polar Lipids) after multiple liquid nitrogen freeze/thaw cycles, and purified over a PD-10 desalting column (GE Healthcare). 3 μg of BHA were bound to GD_1a liposomes in liposome buffer and the solution was acidified from a pH range of pH 6.25 to pH 5.0 for 4500 sec post-acidification. The BHA-mediated SRB dequenching was monitored at 22 °C with excitation/emission at 565/585 nm on a Varian Cary Eclipse Fluorescence Spectrophotometer. Reported fluorescence intensities were normalized at a given time point after acidification, relative to an average non-acidified time point, and an averaged maximally dequenched intensity, generated upon liposome disruption with Triton-X100 (Sigma-Aldrich).

Garcia N.K., Guttman M., Ebner J.L, & Lee K.K. (2015). Dynamic changes during acid-induced activation of influenza hemagglutinin. Structure (London, England : 1993), 23(4), 665-676.

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Lipid Composition Mimicking S. citri Membrane

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All the lipids used in the experiments were purchased from Avanti Polar Lipids, namely, DOPC (850375C), DOPG (840475C), porcine brain sphingomyelin (860062C), and Cardiolipin (710335C). For performing pelleting assays with liposomes mimicking S. citri lipid composition, the following protocol (earlier reported in Harne et al., 2020 (link)) was used. A stock concentration of 2 mM lipids in chloroform was made for S. citri membrane mimic, only DOPG, only DOPC, and varying percentage ratios of DOPC:DOPG lipids. Chloroform solution of lipids was aliquoted in a clean test tube and dried. Dried lipids were resuspended in buffer A (50 mM Tris, pH 8, and 300 mM KCl) and 1 mM MgCl₂. This lipid solution was extruded through a 100-nm polycarbonate membrane (Avanti Polar Lipids) to get liposomes of 100-nm range. These liposomes were further used in the charge specificity and nucleotide-dependent liposome pelleting assays.

Pande V., Mitra N., Bagde S.R., Srinivasan R, & Gayathri P. (2022). Filament organization of the bacterial actin MreB is dependent on the nucleotide state. The Journal of Cell Biology, 221(5), e202106092.

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Liposome Formulation with Ni-NTA Lipids

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All lipids were purchased from Avanti Polar Lipids (Alabaster, AL). Lipids dissolved in chloroform were mixed, dried down to a thin film, and placed under high vacuum for 2 h. Lipid mixtures included DSPC:DGS-NTA(Ni):Chol (50:30:20), DSPC:DGS-NTA(Ni):Chol (70:10:20), and DSPC:DGS-NTA(Ni):Chol (75:5:20). The films were re-hydrated with 1 mL PBS at 60°C and vortexed. The liposomes were sonicated at 60°C for 10 min. The liposomes were extruded through a 100 nm polycarbonate membranes (Avanti Polar Lipids) at 60°C. Liposomes were incubated with a 660:1 molar ratio of DGS-NTA(Ni) lipid to His-tagged 58C-scFv to ensure maximum binding at 4°C overnight. A sepharose CL-6B size exclusion column was used to remove free, unbound 58C-scFv. Fluorescently labeled 58C-scFv was used to determine the amount of scFv bound to liposomes. The low valency formulation had approximately 14 58C-scFvs per liposome, the medium valency formulation had approximately 28 58C-scFvs per liposome, and the high valency formulation had approximately 84 58C-scFvs per liposome (Supplemental Table 1).

Deci M.B., Ferguson S.W., Scatigno S.L, & Nguyen J. (2018). Modulating macrophage polarization through CCR2 inhibition and multivalent engagement. Molecular pharmaceutics, 15(7), 2721-2731.

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PLGA Nanoparticle Characterization and Cell Assays

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The PLGA were purchased from LACTEL Absorbable Polymers (Brimingham, USA). The 100 nm polycarbonate membranes were purchased from Avanti Polar Lipids (Alabama, USA). The GST-ETX monoclonal antibody was developed previously by colleagues in our laboratory. MDCK cells were preserved previously by colleagues in our laboratory. Mouse neuroblastoma N2a cells were purchased from BeNa Culture Collection (Beijing, China). The MTS were purchased from Promega Corporation (Madison, USA). BABL/c mice of SPF grade were purchased from Sipeifu (Beijing, China). Cy5.5-antibody conjugation kits were purchased from Bioss (Beijing, China). DiR were purchased from Invitrogen (Carlsbad, USA).

Xu J., Li D., Kang L., Liu T., Huang J., Li J., Lv J., Wang J., Gao S., Li Y., Yuan B., Zhao B., Wang J, & Xin W. (2023). Systematic evaluation of membrane-camouflaged nanoparticles in neutralizing Clostridium perfringens ε-toxin. Journal of Nanobiotechnology, 21, 95.

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Lipid Bilayer Preparation and Functionalization

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BPLE (141101; Avanti Polar Lipids, Alabaster, AL) or Folch lipids (B1502; Sigma Aldrich, St. Louis, MO) were used as is. Lipid stocks (Avanti Polar Lipids) were aliquoted in the following proportions to generate PIP₂-, NTA-, or PIP₂ plus NTA–containing mixtures: DOPC:DOPS:DOPIP₂ (84:15:1 mol%), DOPC:DOPS:NTA (80:15:5 mol%), and DOPC:DOPS:DOPIP₂:NTA (79:15:1:5 mol%). When necessary, trace amounts of the fluorescent lipid probe p-Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (DHPE) was incorporated to a final concentration of 1 mol%. For liposomes, dried lipid mixtures were hydrated in assay buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.5, 150 mM KCl) to a final concentration of 1 mM and extruded through 100-nm polycarbonate membranes (Avanti Polar Lipids). SUPER templates were prepared with PIP₂- or NTA-containing liposomes as previously reported (Pucadyil and Schmid, 2010 (link)). SMrT templates were prepared as described previously (Dar et al., 2015 (link), 2017 ).

Dar S, & Pucadyil T.J. (2017). The pleckstrin-homology domain of dynamin is dispensable for membrane constriction and fission. Molecular Biology of the Cell, 28(1), 152-160.

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Preparation of Liposome Mimicking S. citri Membrane

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All the lipids used for liposome preparation were purchased from Avanti Polar Lipids, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 850375C), 1,2-dioleoyl-sn-glycero-3-phospho-(1 0 -rac-glycerol) (DOPG, 840475C), Brain SM (Sphingomyelin (Brain, Porcine), 860062C), E. coli Cardiolipin (841199C). To obtain a final working concentration of the liposome for the assay to 2 mM, 0.28 mM of DOPC, 0.76 mM of DOPG, 0.66 mM of Brain SM and 0.30 mM of E. coli CL chloroform solutions were aliquoted in a clean test tube and dehydrated to remove the chloroform. The concentrations of these liposome were chosen based on the available molar ratio of membrane lipids found in S. citri [72] . For preparing the liposome, dried lipid mixtures were hydrated in buffer A (300 mM KCl, 50 mM Tris, pH 8.0) containing 1 mM MgCl 2 to a final concentration of 2 mM. This mixture was extruded through 100 nm polycarbonate membranes (Avanti Polar Lipids).

Harne S., Duret S., Pande V., Bapat M., Béven L, & Gayathri P. (2020). MreB5 Is a Determinant of Rod-to-Helical Transition in the Cell-Wall-less Bacterium Spiroplasma. Current biology : CB, 30(23).

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