Isolation of Outer Membrane Protein from P. aeruginosa With Octyl-POE

Reference:

Methods in Enzymology, Vol. 125: 309-328, 1986.

Octyl-POE was obtained from Bachem.

Method:

1. Harvest cells at 7K for 15 minutes. Resuspend the pellet in 20% sucrose, 10mM Tris-HCl, DNase I (50ug/ml).
2. French press at 15,000 psi three times. Centrifuge the cell lysate at 3,000 rpm for 10 minutes.
3. Set up a 2-step sucrose gradient (50% and 70%). Apply the sample on top of the gradient and centrifuge at 21,000 rpm overnight in a SW 27 rotor.
4. Collect the outer membrane band by poking a hole at the bottom of the tube and dripping the fraction into a 60 Ti tube.
5. Dilute the fraction with distilled water to less that 20% sucrose. Centrifuge at 45,000 rpm for 1 hour.
6. Resuspend the pellet in 10mM Tris HCl pH 8, 0.5% Octyl-POE. Incubate for 1 hour at 37^oC or sonicate 3 x 10 seconds.
7. Centrifuge at 150,000 g for 1 hour.
8. Retain supernatant and resuspend pellet in 10 mM Tris-HCl pH 8, 3% Octyl-POE. Incubate for 1 hour at 37^oC or sonicate 3 x 10 seconds.
9. Centrifuge at 150,000 g for 1 hour.
10. Repeat steps 8 and 9.
11. Retain supernatant and resuspend pellet in 10 mM Tris-HCl pH 8, 50 mM EDTA, 3% Octyl-POE. Incubate for 1 hour at 37^oC or sonicate 3 x 10 seconds.
12. Centrifuge at 150,000 g for 1 hour.
13. Repeat steps 11 and 12.
14. Resuspend pellet in water.
15. Check all fractions for the protein you seek by SDS-PAGE. Opr P is primarily solubilized in 3% Octyl-POE with 50 mM EDTA.