Osmotic Shock Procedures
Categorized: Outer Membranes
For P. aeruginosa:
- Grow bacteria overnight in 100 ml of the appropriate medium.
- Harvest the bacteria by spinning at 10,000 X g (7,000 rpm in SS34 Sorvall rotor), 4ºC for 10 minutes.
- Resuspend the pellet with 8 ml of 0.05 M Tris-HCl pH 7.3, 0.2 M MgCl. Incubate for 10 minutes at 30ºC.
- Chill in water-ice bath for 15 minutes.
- Warm at 30ºC. for 10 minutes.
- Repeat step 4.
- Pellet shocked cells as in step 2. The supernatant should contain the periplasmic contents. (save both the pellet and the supernatant to run on gels)
- Concentrate the supernatant 10 fold by using either a 10,000 Mol wt cutoff Centricon filter or an Amicon filtration apparatus with a PM10 filter.
- Dialyse concentrated supernatant versus 0.05 M Tris-HCl pH 7.3 to remove any residual MgCl.
- Do Nitrocefin assay to see if shock fluids contain the known standard periplasmic enzyme ß-lactamase.
Neu and Heppel. 1965. J.Biol.Chem. 240:3685; Benz et al. 1978 J. Bact. 135:1080
- Steps 1 and 2 as above.
- Resuspend pellet in 8 ml of 20% w/v sucrose, 0.03 M Tris-HCl pH 8.0. Add 2 ml of 5 mM sodium EDTA (pH 8.0). Mix on rotating shaker at 180 rpm for 10 minutes at room temperature.
- Pellet cells at 4C for 10 minutes at 10,500 rpm in an SS34 Sorvall rotor.
- Resuspend pellet in 10 ml of ice-cold water and mix as before in ice bath for 10 minutes.
- repeat step 4.
- See steps 8-10 above.
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