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

In This Article

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.
1. 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 primarily oxygen in the lines and chamber, providing more consistent bilayer preparations.
2. While purging, stream an inert gas, such as N_2....

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Results

Microfluidic chamber assembly diagram; pipetting, cutting, UV-glue, mounting on coverglass.
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 .......