HighPoint/CannonLab:Lipid Vesicle Preparation

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Current revision

Avanti Polar Lipids has detailed protocols and information on lipids and lipid handling

Liposome Preparation

Lipid Film Preparation

Materials

• CHCl₃
• MeOH
• dry lipids
• screw top glass vial with teflon seal

Procedure

1. Make 8mL of 3:1 CHCl3:MeOH (6mL CHCl3/2mL MeOH)
2. Add 2.5mL of CHCl3:MeOH solution to 25 mg lipids
3. Take 1mL (10 mg lipid) of lipid/CHCl3:MeOH solution and place in 4mL vial (2 dram?)
4. 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
5. Place vial in vacuum desicator overnight to remove remainder of solvent
6. 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
    1. add 1 mL buffer to 10 mg lipid
    2. tape vial to vortexer and shake for 1 h
    3. store hydrated lipids in freezer until you are ready to make LUVs

Large Unilamellar Vesicle Extrusion

Read through the following links on the Avanti Polar Lipids website

• Liposomes

• LUVET

Follow these protocols carefully and you will be successful at producing LUV