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Preparation of liposomes for Biacore assays

Lipids for attachment to Sensor Chip HPA or Sensor Chip L1 are prepared as either large or small unilamellar vesicles (LUV and SUV respectively). The choice of vesicle type is largely a matter of preference in lipid handling, although LUV can in some cases give more stable sensor surfaces while SUV can adsorb more rapidly to the surface.

Vesicles are prepared from stock lipids in two steps:

1. Preparation of multilamellar vesicles (MLV)
2. Preparation of LUV by extrusion or SUV by sonication.

Preparing MLV

1. Prepare lipid mixtures as required in chloroform, chloroform:methanol (2:1) (v/v) or 1,1,1-trichloroethane at 10-50 g/l. Dissolved lipids should can be stored below -50°C.
2. Allow the lipid solutions to reach room temperature, then dispense the required volumes (typically 10-100 µl) to glass tubes. Use glass pipettes or Hamilton syringes to dispense lipid solutions.
3. Evaporate the solvent under a stream of nitrogen gas. Adjust the gas stream so that a thin smooth lipid film is formed. Dry the lipid film under reduced pressure for at least 2 h. Dried lipids should be stored under nitrogen at -20°C or below.
4. Hydrate the lipids at room temperature in the desired buffer (recommended maximum lipid concentration 10 mM). Proteins or other additional components required in the lipid layer can be added at this stage.

Add the buffer to the lipid film, allow to stand for 10 minutes, then shake briefly. Repeat the shaking at 10 min intervals for at least 40 min. Check the preparation visually: it should be a homogenous, turbid emulsion with no visible particles or lipid remnants on the tube wall.

Preparing LUV by extrusion

LUV can be prepared by repeated extrusion of MLV through a small-pore membrane. The size of vesicles obtained is determined largely by the pore size. For best results, extrusion should be performed at 5-10°C above the main transition temperature (Tm) of the lipids.

A lipid extruder is a simple device consisting of two reciprocal glass syringes on either side of a filter membrane. The lipid preparation should always be passed through the extruder an odd number of times so that larger aggregates remain on the starting side of the membrane and do not contaminate the preparation.

1. Pass the MLV preparation 19 times through double Nucleopore polycarbonate filters with pore diameter 50-200 nm.
2. Wash the equipment carefully after use or when changing to a preparation with a different lipid composition.
   For preparations containing lipids only, use the following wash sequence:
       3 times with water
       3 times with 99% ethanol
       3 times with water
   If the preparation includes proteins, wash as above followed by
       once with 1 M HNO3
       5 times with water
   Note: Do not wash with nitric acid directly after ethanol.

Preparing SUV by sonication

1. Heat the water in the sonication bath to 5-10°C above the main transition temperature (Tm) of the lipids.
2. Submerge a tube containing the MLV preparation in the middle of the sonication bath. Make sure the level of solution in the tube is below the water level in the bath.
3. Start the sonication. Check the appearance every 10 minutes, and continue until the preparation is completely clear.
4. Centrifuge the preparation 100,000 g for 1h to remove particles and aggregates. Collect the supernatant.

Storing liposome preparations

Store all LUV and SUV preparations at +4° C. Equilibrate to the experiment temperature just before use. Vesicles should normally be prepared fresh each day, especially if lipids with unsaturated acyl chains are included.

If vesicles are prepared and stored under nitrogen, shelf life may be extended to a few days.

See also

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