Aug 25, 2023
Generation of membrane tubules by lipid-covered silica beads rolling
- Javier Espadas1,
- Aurelien Roux1
- 1Department of Biochemistry, University of Geneva, Geneva, 1211, Switzerland
Protocol Citation: Javier Espadas, Aurelien Roux 2023. Generation of membrane tubules by lipid-covered silica beads rolling. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldpoe7l5b/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: June 02, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82811
Keywords: membrane tubules, lipid-covered silica beads, ASAPCRN, generation of membrane tubule, membrane tubules from lipid, membrane tubule, covered silica bead, silica bead, lipid
Abstract
This protocol explains the high throughput methodology to generate membrane tubules from lipid-covered silica beads.
Attachments
733-1846.docx
16KB
Materials
Materials:
- Lipids:
1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1, 18:1 PC)Avanti Polar Lipids, Inc.Catalog #850375P
12-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt)Avanti Polar Lipids, Inc.Catalog #840035
12-Dioleoyl-sn-glycero-3-phosphoethanolamine labeled with Atto 647NMerck MilliporeSigma (Sigma-Aldrich)Catalog #42247
- Glass vials (2700 Supelco, Sigma-Aldrich).
- Silica Beads Microspheres-NanospheresCatalog #140256-10
- Parafilm.
- Petri dish.
- ChloroformMerck MilliporeSigma (Sigma-Aldrich)Catalog #650498
- sticky-Slide VI 0.4IbidiCatalog #80608
- Bovine serum albumin 2 mg/mlThermo Fisher ScientificCatalog #23209
Solutions:
- Lipid films hydration buffer A: 25 mM HEPES 7.4.
- Working buffer:
| A | |
| 20mM HEPES 7.4 | |
| 150mM NaCl | |
| 2.5mM MgCl2 | |
| 5% Glycerol | |
| 2mM DTT |
Protocol
3h 15m
Mix of DOPC, DOPS and Atto 647N DOPE at 59.9:40:0.1 mol% respectively in a final volume of 200 µL with chloroform and 0.5 g/L lipid final concentration in a glass vial.
Dry the lipid mixture in the glass vials for 02:00:00 in a vacuum chamber forming the dried lipid films on the bottom of the glass vials.
2h
Add 200 µL of the lipid films hydration buffer A to the glass vial containing the dried lipid films.
Vortex the glass vials until visually seeing complete resuspension of the dried lipid films in the solution (seen by an increase in the turbidity of the lipid solution) forming the multilamellar vesicles (MLVs).
Mix 10 µL of MLVs with 2 µL of silica beads in an Eppendorf.
Deposit 6 drops of 2 µL each containing the mixture of MLVs and silica beads on a parafilm slide placed in the bottom of a petri dish.
Dry the drops for 01:00:00 in the vacuum chamber until the liquid is completely dried.
1h
Stick a microfluidic device on a 1.5 borosilicate coverslip.
Passivate the microfluidic channels by adding 200 µL solution of 2 g/L BSA for 00:15:00 .
15m
Clean the BSA solution by passing 200 µL working buffer solution in each channel 5 times.
Add 200 µL of working buffer to each channel with a final concentration of GFP-LRRK2 of 500 nanomolar (nM) .
Pick one of the dried drops and add it to the inlet of the microfluidic device.
Gently tilt the chamber towards you 60 degrees with the inlet in the upper part and the outlet in the lower part, and wait until visually seeing the lipid-covered silica beads moving from the inlet to the outlet openings of the microfluidic device.
Wait until reaching the steady state of protein coverage on the lipid tubules.