HighPoint/CannonLab:Lipid Vesicle Preparation
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Avanti Polar Lipids has detailed protocols and information on lipids and lipid handling
Lipid Film Preparation
Materials
- CHCl3
- MeOH
- dry lipids
- screw top glass vial with teflon seal
Procedure
- Make 8mL of 3:1 CHCl3:MeOH (6mL CHCl3/2mL MeOH)
- Add 2.5mL of CHCl3:MeOH solution to 25 mg lipids
- Take 1mL (10 mg lipid) of lipid/CHCl3:MeOH solution and place in 4mL vial (2 dram?)
- Dry using rotovap
- Attach vial to rotovap with a rubber septum (silicone septa only work 2-3 times before shrinking)
- Insert needle through septum to allow solvent to evaporate
- Evaporation takes <<30 min if conditions are right
- Place vial in vacuum desicator overnight to remove remainder of solvent
- Close tightly and store aliquot in freezer
Lipid Film Hydration
- Prepare the buffer appropriate for your experiments
- Determine the concentration of lipids you wish to use in your experiments
- Prepare a hydrated lipid suspension much more concentrated than the desired experimental concentration
- A concentration of 10 mg/mL is often a good starting point
- add 1 mL buffer to 10 mg lipid
- tape vial to vortexer and shake for 1 h
- store hydrated lipids in freezer until you are ready to make LUVs
- A concentration of 10 mg/mL is often a good starting point
Large Unilamellar Vesicle Extrusion
Read through the following links on the Avanti Polar Lipids website
Follow these protocols carefully and you will be successful at producing LUV