The vesicles were passed through a 50 nm polycarbonate membrane (GE Healthcare, Chicago, IL, USA) fitted on an extruder (Avanti Polar Lipids, Alabaster, AL, USA). Vesicles before and after extrusion were analyzed for vesicle size and deformability index. Vesicle size was analyzed following the procedure described in Measurement of Vesicle Size. The deformability index was calculated by dividing vesicle size before and after extrusion.30 (link)
50 nm polycarbonate membrane
Manufactured by GE Healthcare
Sourced in United States
The 50 nm polycarbonate membrane is a size-selective filtration material designed for laboratory applications. It features a pore size of 50 nanometers, which allows the passage of small molecules and particles while retaining larger components. This membrane is composed of polycarbonate, a durable and chemically resistant material suitable for various laboratory procedures.
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Lab products found in correlation
3 protocols using 50 nm polycarbonate membrane
1
Extrusion of Lipid Vesicles for Size and Deformability
2
Supported Lipid Bilayer Formation and CD59-ILY Interaction
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For the preparation of supported lipid bilayers, pure lipids were dissolved in chloroform at 10 mg/mL and mixed in solution to give a lipid mixture of DOPC:cholesterol 2:1 molar ratio. The lipid-in-chloroform solution was then dried in a glass vial under a stream of nitrogen gas to give 1 mg of lipid as a thin film. The lipid film was hydrated in buffer (20 mM Tris, 200 mM NaCl, pH 7.5), vortexed and bath sonicated to give a cloudy lipid suspension. The suspension was then passed through a 50 nm polycarbonate membrane (GE Healthcare Lifesciences) 15 times to yield a clear suspension of small unilamellar vesicles.
Supported lipid bilayers were formed by injecting 4.5 µL of the lipid vesicle suspension to a freshly cleaved mica disk (6 mm diameter) under 18 µL of incubation buffer (hydration buffer plus 10 mM CaCl2 solution) at 37 °C; this induces the rupture of the vesicles onto the mica support over an incubation period of approximately 30 min. Excess vesicles were then removed from the supernatant by rinsing with 500 µL of the hydration buffer, to yield a uniform bilayer free of adsorbed vesicles (as assessed by AFM imaging). The supported lipid bilayers were next incubated with a final concentration of 50 ng/ml CD59 for 5 min, and thereafter with a final concentration of 100 µg/ml ILY for 15 min, all at 37 °C, and next washed with 500 µL of the hydration buffer.
Supported lipid bilayers were formed by injecting 4.5 µL of the lipid vesicle suspension to a freshly cleaved mica disk (6 mm diameter) under 18 µL of incubation buffer (hydration buffer plus 10 mM CaCl2 solution) at 37 °C; this induces the rupture of the vesicles onto the mica support over an incubation period of approximately 30 min. Excess vesicles were then removed from the supernatant by rinsing with 500 µL of the hydration buffer, to yield a uniform bilayer free of adsorbed vesicles (as assessed by AFM imaging). The supported lipid bilayers were next incubated with a final concentration of 50 ng/ml CD59 for 5 min, and thereafter with a final concentration of 100 µg/ml ILY for 15 min, all at 37 °C, and next washed with 500 µL of the hydration buffer.
3
Preparation of Small Unilamellar Lipid Vesicles
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Pure lipids were dissolved in chloroform at 10 mg/mL and mixed in solution to give a lipid mixture at a desired molar ratio. The lipid-in-chloroform solution was then dried in a glass vial under a stream of nitrogen gas to give 1 mg of lipid as a thin film. The lipid film was hydrated in buffer (20 mM HEPES, 120 mM NaCl, pH 7.4), vortexed and bath sonicated to give a cloudy lipid suspension. The suspension was then passed through a 50 nm polycarbonate membrane (GE Healthcare Lifesciences) 15 times to yield a clear suspension of small unilamellar vesicles (SUVs). All lipid species used had a gel-to-fluid transition below room temperature, and therefore were assumed to be miscible without heating.
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