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

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.

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