Liposome and Lipid tubule Production
Protocol From Harvey McMahonÝs lab
To make lipid tubes instead of vesicles, substitute in galactocerebrosides (Sigma C 1516) for the PE, up to 40% GalCer: e.g., 10% Chol, 40 % GalCer, 40% PC 10% PIP2. These lipid tubes will have a diameter of approximately 40 nm. To make Folch liposomes, simply use folch extract straight (10 mg/ml in 19:1 ChCl3/MeOH) instead of mixing lipids.
See also advice on making Liposomes from Avanti Polar Lipids.
1. Rinse 1.8 ml glass vials (with teflon caps, Wheaton #224740) in chloroform to wash.
1. Mix 100Ál of chloroform (special stuff) and 30Ál of methanol. Dry chloroform, stored with dessicant, is recommended (e.g. Sigma C-2432)
2. Add lipids (most important last) eg. 10% cholesterol (stock: 10mg/ml), 35% PC (stock: 10mg/ml), 35% PE (stock: 10mg/ml), 10% PS (stock: 20mg/ml), 10% PIP2 (stock: 1mg/ml). Lipids are already in chloroform, keep them on ice. The concentration of the final mixture is 1mg/ml. Gently swirl in-between additions. Be sure to use protonated PIP2 (see protocol, or use the pre-protonated ones from Avanti Polar Lipids), mix them first or sonicate in the bath sonicator.
3. Evaporate chloroform + methanol using slow-flow Argon (0.1 units) to produce a film on the glass. When this film becomes white, turn up the flow and spread out last drops. Avoid leaving thick lumps of precipitated lipid.
4. Put in a dessicator, pump out the air, leave for 5-15 minutes.
5. Slowly release the valve.
6. Add 1ml of filtered buffer. Leave to hydrate for 5 minutes at room temp. Gently agitate occasionally.
7. Put in a bath sonicator for about 30 seconds to 2 minutes to resuspend the lipids and form liposomes/tubes, until solution just begins to clear.
8. Apply two to five brief pulses with a small (2 mm) probe sonicator. Solution should be only slightly cloudy.
9. Filter using 0.1, 0.2, or 0.4Ám polycarbonate filters, e.g., Whatman Cyclopore Cat # 7060 2501, with 1 ml syringes and Avanti mini-extruder. Push 3-11 times through the filter (odd number so that the material trapped in the filter does not get washed back into the final vial) and deposit into a buffer-rinsed glass vial. NB: the filtration process usually leads to loss of around 100-200 ul of liquid, and 10-20% of the lipid in the reamining volume.
NB. Galactocerebrosides and PIP2 may need to be left on the bench for a few minutes to warm up since they are insoluble at low temperatures.
PIP2 = PtdIns(4,5)P2
Folch extract= Total brain lipids from Sigma (B-1502), approx. 10% phosphoinositides.