Outer Membrane Preparation - One Step Sucrose Gradient Procedure

1. Grow 2 litres of cells OD₆₀₀ = 0.8-1.0, pellet by centrifugation in the GS3 rotor for 10 minutes at 7000rpm and resuspend in 20ml cold 20% sucrose in 10 mM Tris pH 8.0, 50 µg/ml DNase I. Incubate at least 15 minutes at room temperature before french pressing.
   
   NOTE: The cells may be frozen in 20% sucrose to enhance breakage. The 20% sucrose plasmolyses cells, resulting in better separation of the outer and inner membranes.
   
2. Break twice at 15,000 psi in the large French Pressure cell and put broken cells on ice immediately to stop protease action.
   
3. Remove the cell debris by centrifugation at 3,000 rpm for 10 minutes at 4ºC.
   
4. A one-step sucrose gradient is used. The sucrose concentrations in the steps off the gradient may also be varied depending on the degree of separation desired. Use Beckman polyallomer tubes for the gradients.
   
5. For crude separation of outer and inner membranes, use a
   2-step gradient:
   
   

   For the SW 41 rotor	For the Sw 27/8 rotor
   4 ml 70% sucrose	14 ml 70% sucrose
   4 ml 60% sucrose	14 ml 60% sucrose
   4 ml sample (in 20% sucrose)	9 ml sample (in 20% sucrose)
   Adjust the volume of the 70% cushion to fill the tube.	To increased yield of outer membranes use 50% sucrose in instead of 60% sucrose

   
   
6. Centrifuge for 4 hours or overnight at 39,000 rpm in the SW 41 rotor at 4^oC. Centrifuge overnight at 23,000 rpm in the SW 27/8 rotor at 4^oC.
   
7. There should be two bands for the 2-step gradient. The upper band should be reddish and the lower one should be white. The lower band is the outer membrane. Outer membranes will band at the junction between the 60% and 70% steps. Inner membranes will band at the junction between the 20% and 60% steps. Collect the membranes by poking a hole in the bottom of the tube with a hot needle, and let the sucrose drip out, collecting only the membrane band.
   
8. Collect the bands separately using the bottom-drip method.
   
9. Place each band in a 60Ti tube and add at least two volumes of distilled water to dilute the sucrose below 20%.
   
10. Centrifuge at 47,000 rpm for 1 hour.
    
11. Using a syringe, resuspend the pellets in approximately 1ml.