Planar Supported Lipid Bilayer Assay: A Microscopy-Based In Vitro Technique to Investigate Septin Assembly on Biological Membrane Mimics

Published: April 30, 2023

Abstract

Source: Curtis, B. N. et al. Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions. J. Vis. Exp. (2022)

In this video, we demonstrate a procedure to investigate septin organization on a glass-supported lipid bilayer in vitro using total internal reflection fluorescence microscopy.

Protocol

1. Planar Lipid Bilayers Plasma Cleaning of the Slides NOTE: Plasma cleaning removes organic contaminants and increases the hydrophilicity of the glass, ensuring efficient lipid adsorption. The following steps may change or need to be optimized depending on the plasma cleaner and glass coverslips used. Purge the plasma cleaner for 5 min with oxygen to remove air from the lines and chamber. This is to ensure that, when the plasma cleaner is run, there is pri…

Representative Results

Figure 1: Schematic of reaction chamber set up. To prepare custom chambers, a 0.2 mL PCR tube is cut where the tube begins to taper and at the cap (red dashed lines). The uncut rim of the cut tube is then coated with a thin layer of UV-activated glue (blue) and placed glue-down on a coverslip.

Disclosures

The authors have nothing to disclose.

Materials

0.2 mL PCR Tubes with flat cap, Natural Watson 137-211C(EX)
HEPES Sigma Aldrich H3375-1KG
Magnesium chloride VWR 7791-18-6
Methyl cellulose 4000cp Sigma-Aldrich M052-100G
Microglass coverslips for planar bilayers Matsunami Discontinued 22×22
Optical Adhesive Norland Thorlabs NOA 68 Flexible adhesive for glass or plastics
Plasma Cleaner Plasma Etch PE-25 Voltage: 120V, 60Hz. Current: 15 AMPS
Potassium chloride VWR 0395-1kg
UV Lamp Spectroline ENF-260C 115 Volts, 60 Hz, 0.20 AMPS
Bovine Serum Albumin (BSA) Sigma-Aldrich A4612-25G
Beta mercaptoethanol (BME) Sigma-Aldrich M6250-100ML
DOPC Avanti Polar Lipids 850375
Egg Liss Rhodamine PE Avanti Polar Lipids 810146

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Cite This Article
Planar Supported Lipid Bilayer Assay: A Microscopy-Based In Vitro Technique to Investigate Septin Assembly on Biological Membrane Mimics. J. Vis. Exp. (Pending Publication), e21163, doi: (2023).

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