Last updated: 2013 July 02
<Authors:Caroline Ajo-Franklin. Note: This procedure was developed in Steve Boxer's laboratory. Key contributors were Li Kung, Jennifer Hovis, and Chiaki Treynor.
Small unilamellar vesicles can be formed by extruding aqueous lipid solutions through membranes with small pores. These vesicles can be used to reconstitute membrane proteins or to make supported bilayers.
- Lipids (usually egg PC, DOPC, or POPC from Avanti Polar Lipids)
- optional: fluorescently labeled lipids, e.g. Texas Red DHPE Invitrogen #T-1395, note: these are present in the -20C common stocks
- optional: buffered solution of water-soluble fluorescent dye to be encapsulated, e.g. 20 mM 5,6-carboxyfluorescein in 10 mM Tris equilibrated with 50 mM NaOH to pH 8, plus 65 mM NaCl or (HPTS, add details), and Sepharose CL-6B size exclusion media (catalog # X)
- vesicle reconstitution buffer, typically 10 mM Tris, 100 mM NaCl, alternatively Millipore water
- 7.5 mL glass vials with phenolic caps for lipid storage
- 20 mL scintillation vials for use with the Biotage V-10 evaporator (VWR catalog #66022-128)
- Millipore water
- absolute ethanol, note: this should not be denatured EtOH
- Hamilton syringes 25 uL, 100 uL, 500 uL (VWR catalog#: 60372-105, 60373-040. 60373-083)
- Mini-extruder (Avanti Polar Lipids # 610000)
- filter supports (Avanti Polar Lipids # 610014)
- polycarbonate membranes (Avanti Polar Lipids #610014)
- clean dessicator (must be free from desiccant)
- Biotage V-10 Evaporator (see equipment manager for training)
Important Safety Concerns:
- CHLOROFORM is dangerous! May be fatal is swallowed, inhaled, or absorbed through skin. Also has potential to affect the central nervous system, cardiovascular system, liver and kidneys. You must wear the gloves, safety glasses, and lab coat while working with this solvent. All chloroform work should be done in a fume hood.
- The glass syringes used to contain the vesicle solution during extrusion are fragile, and can break easily if mishandled or dropped. If the glass breaks or cracks, dispose of the syringes in a broken glass container in the lab. A broken glass container can be found in room 5204. The blunt tip needles on the syringe are not sharp, but can still puncture a glove or skin if not handled carefully.
- Be sure to contact the equipment manager for training on the Biotage and TIRF microscope!
Waste Disposal & Clean-up:
- Dispose of excess chloroform in the red flam cans in 5209.
- Dispose of glass vials in the broken glass containers.
- Lipid solutions can be poured down the drain, unless they contain dyes. Then the solution should be disposed the liquid biohazard water containers and logged. Unused buffer can be disposed in the drain.
- Decide the composition of the vesicles. First, 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. Second, some lipids such as DOPS do not form vesicles or do not form supported bilayers. Third, lipids have different head groups with varying charges which can be a critical aspect for membrane protein insertion. You can mix different types of lipids to have a vesicle that might resemble the composition of a protein’s native environment. Fourth, if you would like to visualize the vesicles or bilayers, you need to incorporate a lipid conjugated with a fluorescent head group, Texas-Red- DHPE for example.
- Lipids must be completely dried of all chloroform before they are resuspended in a desired buffer. Proper vesicles will not form in the presence of the organic solvent chloroform.
- Protect the dye from light, so the fluorescence is not photo-bleached prior to imaging. Wrap Texas-Red DHPE vials in aluminum foil or aliquot in glass vials which have the dark colored coating to reflect light. Also wrap the lipid/dye dried mixture in aluminum before setting to dry.
- Use vesicles promptly. They are typically able to form supported bilayers for ~1 week after extrusion or reconstitution.
- 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.
- Determine the concentration of final lipid solution and calculate volume of each type of lipid required. Typically, we use ~5mg of lipid per vesicle preparation.
- If needed, use chloroform to re-dissolve dried aliquots of lipids.
- Add appropriate volumes of lipid stock into a clean 20 mL scintillation vial. When transferring the stock solution, use syringes. Do not use pipetmen as i) they will be damaged by the use of organic solvents and ii) the low viscosity of chloroform will cause solution to drip out of tips and inaccurate measurements.
- Use the Biotage V-10 to evaporate the chloroform.
- Dry remaining chloroform residue by placing the lipid vial in a vacuum chamber for at least 1-2 hrs or overnight.
- Rehydrate the lipids in buffer, Millipore water, or the buffered water-soluble dye solution to ~10 mg/mL. Vortex the solution to redissolve as much lipid material as possible. The lipid/aqueous solution should result appear cloudy. Allow lipids to rehydrate for 15 min before extruding.
- Meanwhile, 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 or at room temperature if the gel phase transition temperature is greater than 4ºC.
- Disassemble the extruder. Dispose of the polycarbonate membranes and filter supports as chemically-contaminated solid waste (on the bio-side, this means the white bins).
- Remove the o-rings from the teflon holders. Rinse all components of the extruder with Millipore water twice and absolute ethanol twice. Allow the components to dry on the Kimwipes, then return to storage.
- Optional for dye-encapsulated vesicles:
- Pack a gravity column with Sepharose CL-6B. A 15 mm diameter x 70 mm long column (10 mL bed volume, ThermoScientific catalog # 29925) works well for separating vesicles from non-encapsulated dye.
- Equilibrate the column with at 3 bed volumes of iso-osmotic buffer.
- Gently load less than 1 mL of the vesicle solution into the column. Allow it to enter the column, so the meniscus just touches the top of the size-exclusion media.
- Add additional buffer to the column in 1 mL aliquots.
- Collect fractions as they elute from column tip. The vesicle containing fraction will appear somewhat whiteish (if no fluorescent lipids are present) or colored if fluorescent lipids are present.
- Wash the column extensively with at least 5 column volumes of buffer to remove any non-encapsulated dye from the column.
- Verify the contents of the fractions using UV-Vis spectroscopy.