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).