Hancock Lab

Osmotic Shock Procedures

Categorized: Outer Membranes

Reference:

Poole and Hancock. l984. Eur.J.Biochem.144:607-612.

Method:

For P. aeruginosa:

  1. Grow bacteria overnight in 100 ml of the appropriate medium.
  2. Harvest the bacteria by spinning at 10,000 X g (7,000 rpm in SS34 Sorvall rotor), 4ºC for 10 minutes.
  3. 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.
  4. Chill in water-ice bath for 15 minutes.
  5. Warm at 30ºC. for 10 minutes.
  6. Repeat step 4.
  7. 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)
  8. 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.
  9. Dialyse concentrated supernatant versus 0.05 M Tris-HCl pH 7.3 to remove any residual MgCl.
  10. Do Nitrocefin assay to see if shock fluids contain the known standard periplasmic enzyme ß-lactamase.

For E.coli:
Neu and Heppel. 1965. J.Biol.Chem. 240:3685; Benz et al. 1978 J. Bact. 135:1080

  1. Steps 1 and 2 as above.
  2. 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.
  3. Pellet cells at 4C for 10 minutes at 10,500 rpm in an SS34 Sorvall rotor.
  4. Resuspend pellet in 10 ml of ice-cold water and mix as before in ice bath for 10 minutes.
  5. repeat step 4.
  6. See steps 8-10 above.

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