NanoBio: Making Unilamellar Vesicles
|Line 51:||Line 51:|
This procedure was developed in Steve
This procedure was developed in Steve laboratory. Key contributors were Li Kung, Jennifer Hovis, and Chiaki Treynor.
Revision as of 15:48, 28 August 2007
PREPARATION OF SMALL UNILAMELLAR VESICLES
This procedure details how small (~35-100 nm diameter) unilamellar vesicles can be formed by extrusion.
- Lipids (usually egg PC and 1-2 mole % dye)
- Wide-mouth screw top glass vials, ~ 10 mL
- ICN 7x detergent, i.e MP Biomedicals catalog # 097667093
- Millipore water
- Absolute ethanol
- 50 uL Hamilton syringe, to be used for lipids only
- 250 uL Hamilton syringe, to be used for lipids only
- Hot plate
- Extruder, e.g. mini-extruder from Avanti Polar Lipids
- Filter supports
- Polycarbonate membranes
- Clean dessicator free from desiccant
- Clean glass vials. Immerse vials in 20-30% 7x detergent (70-80% distilled water) that has been heated until clear (~90ºC) for 5 minutes. Rinse thoroughly with double distilled water. Rinse three times with acetone, then three times with chloroform. Dry with N2(g).
- Add appropriate quantities of lipids in chloroform to clean glass vials. It is recommended that you keep a set of syringes to be used only for lipids.
- Dry the lipids down with N2(g). Ideally, the lipids will form a thin uniform film over much of the interior glass surface.
- Dry the lipids under vacuum for 1 hour. If this is done in a desiccator, do not use desiccant.
- Rehydrate the lipids in buffer or Millipore water to ~10 mg/mL. Vortex the solution to redissolve as much lipid material as possible. Allow lipids to rehydrate for 15 min before extruding.
- Clean the components of extruder. Rinse pieces with absolute ethanol, allow to dry on Kimwipes, and repeat.
- Assemble the extruder with filter supports (4 total) and polycarbonate membranes (2 total). See extruder instructions for more details.
- Rinse the assembled extruder with Millipore water three times to remove any air bubbles.
- Pass the lipid solution ~20 times through the polycarbonate membranes. You should feel slight resistance when pushing the syringes, and the lipid solution should become transparent (vs. opaque).
- Store the vesicles at 4ºC.
- Rinse the disassembled extruder first with water, then with ethanol. Allow to dry on Kimwipes.
- Cleanliness of the glass vials is very important. It is crucial that there is no visible aqueous liquid in the glass vials before addition of the lipids in chloroform.
- Having a uniform film of lipids on the interior of the glass vial makes rehydration of the lipids easier.
- The inability to form a uniform lipid film can be an indication that the glass vial is not sufficiently clean.
- Sometimes excessive pressure can build up in the extruder, and passing the lipid solution through the membrane requires a great deal of force. When this occurs, remove the lipid solution from the syringe, re-assemble of the extruder with fresh filter supports and polycarbonate membranes, and re-extrude the lipid solution.
- If the lipid solution does not become clear and/or it requires very, very little force to pass the lipid solution through the extruder, then it is possible that the polycarbonate membranes have developed a tear. After completing 20 extrusions, disassemble the polycarbonate and check for a tear. If one is present, re-extrude the lipid solution.
- It is important to use lipid compositions which are in the liquid phase at the temperature which the vesicles are formed. Since egg PC is fluid to ~-10C, vesicles of egg PC can be created at room temperature and stored at 4C. Other compositions may require handling at different temperatures.
This procedure was developed in Steve Boxer's laboratory. Key contributors were Li Kung, Jennifer Hovis, and Chiaki Treynor.
Cremer & Boxer J. Phys. Chem. B 1999
Last updated: Caroline Ajo-Franklin 06 August 2007.