REFERENCES: Nikaido and Roseenberg (1983) J.Bact 153: 241-252; Yoshimura et al. (1983) J.Bact 258: 2308-2314.
METHOD:
- 6.2 µmoles of lipid (ideally egg phophatidylcholine or dipalmitoyl phosphorylcholine) and 0.2 µmole dicetylphosphate are dried at the bottom of a vacuum tube under a stream of nitrogen.
- Suspend lipid film in 0.2 ml aqueous solution of purified porin or porin containing cell envelopes. It is best to use a concentrated protein solution (< 1 mg/ml) so that most of the detergent in the protein solution will be diluted out.
E. coli porins: use 5-20 µg/200 µl P. aeruginosa porins: use 2-10 µg/200 µl
- Sonicate in water bath sonicator on high 15-30 secs. Suspension should turn from opaque to translucent.
- Dry suspension in the same vacuum tubes by warming them in a 45ºC water bath while evacuating using tube connected to the vacuum pump via a CuSO4 drying tube. The drying process takes 2-3 minutes.
- Store liposomes overnight in evacuated dessicator containing CuSO4.
- Gently resuspend liposomes in 0.4 ml of following and leave undisturbed at RT (23ºC) for 2h then gently resuspend by shaking with hand.
12 mM stachyose (E. coli) or 17% w/v Dextran T-20 (P. aeruginosa) 4 mM sodium NAFD ( add NAOH to NAD sol. to pH 6.0) 1 mM imidazole-NAD (pH imidazole to 6.0 with dry NAD
- Filter suspension through 8 um Millipore membrane filter to remove large aggregates.
- Make up 5 mls of sugar solutions:
E coli:18 mM sugar or P aeruginosa: 40 mM sugar 1 mM sodium NAD 1 mM imidazole-NAD pH 6.0 Sugars should range in MW from 150-700.
- Measure liposome swelling by following the change in OD400 using the Perkin-Elmer spectrophotometer and the attached chart recorder. The rate is estimated from the initial slope. To do this, as quickly as possible, add 10 to 20 µl of the liposome suspension to a cuvette containing 0.6 ml of the sugar solution and turning on the chart recorder.
- An important control are liposomes made without protein but with the same amount of detergent as added for the protein-containing liposomes.
