Jul 25, 2022
Membrane Tube Assay
- 1Hurley Lab
Protocol Citation: Liv Jensen 2022. Membrane Tube Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1n3dpgr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: May 03, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 61845
Keywords: Membrane Tube Assay, Imaging buffer, Biotinylated GUVs, ASAPCRN
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Abstract
This protocol details about the Membrane Tube Assay.
Attachments
416-897.pdf
50KB
Materials
Materials:
- Biotinylated GUVs (0.001% mol fraction DSPE-PEG(2000) Biotin, Avanti Polar Lipids), formed by PVA swelling method.
- Small volume flow cells of the type commonly employed for in vitro single molecule imaging (melted parafilm sandwiched between no. 1.5 coverglass)
- Streptavidin functionalized silica beads, 1.56 µm diameter (Spherotech)
- Bovine serum albumin (BSA)
- Confocal fluorescence microscope modified with an optical trap.
- Fluorescently labeled protein
Imaging buffer
( iso- osmotic to GUV swelling solution)
| A | B | |
| Tris pH 8.0 | 20 mM | |
| NaCl | 150 mM | |
| TCEP | 5 mM | |
| MgCl2 | 2 mM |
Membrane Tube Assay
Membrane Tube Assay
Passivate flow cell with 1 mg/mL BSA in imaging buffer.
Rinse flow cell with 2 flow cell volumes of imaging buffer.
Mix GUVs with fluorescent protein and add to flow cell, allowing GUVs to settle on the bottom surface of the flow cell.
Add 1 µL of a 1:1000 dilution of silica beads to flow cell.
Trap a bead in the optical trap, bring into contact with a GUV, and retract, forming a membrane tube.
Visualize protein recruitment to membrane tube with confocal microscopy.