Generation of membrane tubules by lipid-covered silica beads rolling

Mix of DOPC, DOPS and Atto 647N DOPE at 59.9:40:0.1 mol% respectively in a final volume of 200µL with chloroform and 0.5g/L lipid final concentration in a glass vial.

Dry the lipid mixture in the glass vials for 2h 0m 0s in a vacuum chamber forming the dried lipid films on the bottom of the glass vials.

Add 200µL 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 10µL of MLVs with 2µL of silica beads in an Eppendorf.

Deposit 6 drops of 2µL 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 1h 0m 0s in the vacuum chamber until the liquid is completely dried.

Stick a microfluidic device on a 1.5 borosilicate coverslip.

Passivate the microfluidic channels by adding 200µL solution of 2g/L BSA for 0h 15m 0s.

Clean the BSA solution by passing 200µL working buffer solution in each channel 5 times.

Add 200µL of working buffer to each channel with a final concentration of GFP-LRRK2 of 500nanomolar (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.