Aug 25, 2023

Public workspaceGeneration of membrane tubules by lipid-covered silica beads rolling

DOI
dx.doi.org/10.17504/protocols.io.n92ldpoe7l5b/v1
  • Javier Espadas1,
  • Aurelien Roux1
  • 1Department of Biochemistry, University of Geneva, Geneva, 1211, Switzerland
xinbo.wang
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.n92ldpoe7l5b/v1
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
Abstract
This protocol explains the high throughput methodology to generate membrane tubules from lipid-covered silica beads.
Attachments
Icon for 733-1846.docx
733-1846.docx
16KB
Materials
Materials:

  • Lipids:

Reagent1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1, 18:1 PC)Avanti Polar Lipids, Inc.Catalog #850375P

Reagent12-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt)Avanti Polar Lipids, Inc.Catalog #840035

Reagent12-Dioleoyl-sn-glycero-3-phosphoethanolamine labeled with Atto 647NMerck MilliporeSigma (Sigma-Aldrich)Catalog #42247

  • Glass vials (2700 Supelco, Sigma-Aldrich).
  • ReagentSilica Beads Microspheres-NanospheresCatalog #140256-10
  • Parafilm.
  • Petri dish.
  • ReagentChloroformMerck MilliporeSigma (Sigma-Aldrich)Catalog #650498
  • Reagentsticky-Slide VI 0.4IbidiCatalog #80608
  • ReagentBovine 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
Protocol
3h 15m
3h 15m
Mix of DOPC, DOPS and Atto 647N DOPE at 59.9:40:0.1 mol% respectively in a final volume of Amount200 µL with chloroform and Amount0.5 g/L lipid final concentration in a glass vial.
Mix
Dry the lipid mixture in the glass vials for Duration02:00:00 in a vacuum chamber forming the dried lipid films on the bottom of the glass vials.
2h
Add Amount200 µL of the lipid films hydration buffer A to the glass vial containing the dried lipid films.

Pipetting
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 Amount10 µL of MLVs with Amount2 µL of silica beads in an Eppendorf.

Mix
Deposit 6 drops of Amount2 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 Duration01:00:00 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 Amount200 µL solution of Amount2 g/L BSA for Duration00:15:00 .

15m
Clean the BSA solution by passing Amount200 µL working buffer solution in each channel 5 times.

Wash
Add Amount200 µL of working buffer to each channel with a final concentration of GFP-LRRK2 of Concentration500 nanomolar (nM) .

Pipetting
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.