Vesicle Preparation via Extrusion
Purpose
To prepare unilamellar lipid vesicles by extrusion through increasingly smaller filters.
Materials
Description
DLPE
DMPE
DPPE
DSPE
Egg yolk PC
Streptavidin
Texas Red-X-DHPE
Biotin-X-DHPE
Chloroform
Methanol
Supplier
Lipid Type
Avanti
12:0
Avanti
14:0
Avanti
16:0
Avanti
18:0
Avanti
mixed
Boehringer Mannheim
Molecular Probes
Molecular Probes
16:0
Sigma
Aldrich
M.W.
579.75
635.86
691.97
748.07
760.08
52 kDa
1495
1132.61
Tm (˚C)
29
50
63
74
39
Tb=62
Material Notes
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Lipid information: http://www.avantilipids.com
Chloroform dissolves plastic; use only glass or metal syringes and containers for
chloroform solutions.
Sonicate lipid solutions for ~10 minutes before taking aliquots from a stock solution
to break up any aggregates that may be present.
Typically lipid solutions are prepared at 10-20 mg lipid/ml organic solvent, although
higher concentrations may be used if the lipid solubility and mixing are acceptable.
Storage: Minimize exposure to light.
o Powders:
 Saturated lipids: -20˚C
 Unsaturated or tissue derived (e.g. egg PC): Powders are extremely
hygroscopic and quickly absorb moisture. Dissolve powder in organic
solvent and store at -20˚C.
o Aqueous solutions:
 Not recommended for long periods due to resulting hydrolysis of
phospholipids.
Phosphatidylethanolamine (PE): Lipid vesicles containing more than 60 mol% PE
form particles having a small hydration layer surrounding the vesicle. As particles
approach one another there is no hydration repulsion to repel the approaching particle
and the two membranes fall into an energy well where they adhere and form
aggregates. The aggregates settle out of solution as large flocculates that will
disperse on agitation but re-form upon sitting.
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
Egg PC composition: (S/U = 0.88)
Lipid Type
16:0
16:1
18:0
18:1
18:2
20:4
%
34
1.7
11
32
18
3.3
Procedure
Stock Solution Preparation
Lipid (10 mg/ml; 1 ml total):
1. Weigh out 10 mg lipid directly into 1 ml volumetric flask (VWR 29502-022) or
vial with Teflon-coated cap (green).
2. Add 1 ml of a 9:1 (v/v) chloroform/methanol solution (CM) using a glass syringe
with a metal needle.
3. Wrap Teflon tape around glass stopper to seal flask.
4. Store at 4˚C.
Texas Red-X-DHPE (1 mg/ml; 1 ml total):
Note: Try to minimize exposure of fluorophore to light.
1.
2.
3.
4.
5.
Add 1 ml CM to vial to dissolve powder.
Transfer to 1 ml volumetric flask.
Wrap Teflon tape around glass stopper to seal flask.
Wrap flask in aluminum foil to prevent light exposure of flurophore.
Store at 4˚C.
Vesicle Preparation
Prepare Lipid Solution (1 mg/ml lipid + 1 mol% TR; 5 ml total):
1. Calculate appropriate amounts of each component.
2. Combine in 10 ml round bottom flask.
3. Sonicate ~10 minutes.
Dry Lipid:
1. Evaporate solvent off in hood under gas stream while turning flask to coat sides.
2. Attach to vacuum pump with room temperature trap.
3. Wrap in aluminum foil.
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4. Leave vacuum pump on overnight.
Hydrate Lipid:
1. Add 5 ml DI water.
2. Place in oven at a temperature above the gel-liquid crystal transition temperature
(Tc or Tm) of the lipid with the highest Tm.
3. Swirl liquid around. Keep in oven until dye molecules go into solution.
4. Vortex for ~1 minute.
5. Perform 5 freeze-thaw cycles. Freeze solution completely by placing in liquid
nitrogen for ~30 sec; thaw by placing in 60˚C water bath. Wrap wire around top
of vial so that vial can be easily dipped into liquid solutions without exposing
hands. Wear goggles to protect your eyes.
6. Vortex.
Extrude Lipid:
1.
2.
3.
4.
5.
6.
7.
8.
Place two 800 nm filters on top of stainless steel mesh.
Assemble extruder.
Leak test with water.
Extrude lipid solution 3-5 times or until lipid passes through filters quickly (~10
minutes) in Lipex Biomembranes Extruder operated at a temperature above Tm.
Place two 100 nm Nucleopore filters shiny side up on top of the stainless steel
mesh.
Repeat extrusion process 5 times.
Clean extruder with ethanol.
Let extruder parts dry and reassemble.
Vesicle Deposition
1. Cut mica with scissors to the desired dimensions while wearing a mask and
goggles to avoid exposure to mica dust.
2. Dilute the vesicle solution with 0.5 mM KNO3 to a final concentration of 50
g/ml. This subjects the vesicles to an osmotic stress that will aid bilayer
formation.
3. Heat the vesicle solution to just above the lipid Tm.
4. Place a drop of warm vesicle solution onto the bottom of a dish, lay the mica over
the drop, and incubate for 15 minutes at room temperature.
5. Fill the dish with distilled water.
6. Assemble the flow chamber taking care not to expose the membrane-coated
surface to air.
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