Hancock Lab

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

Categorized: Protein Purification and Gel Electrophoresis

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 37oC 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 37oC 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 37oC 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.

 

Method Categories