• Outer Membrane Protein Solubilization from P. aeruginosa with Octyl-POE

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Hancock Laboratory Methods,Department of Microbiology and Immunology,
University of British Columbia, British Columbia, Canada http://www.cmdr.ubc.ca/bobh/showmethod.php?methodid=48 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, 10 mM Tris-HCl, DNase I (50 ?g/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 x 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.

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