Skip to main content

Microsome Preperation

  1. Cells are grown to confluency on P150 dishes.
  2. Place dishes on ice and proceed to do the rest of the protocol on ice. It is possible to process 5 plates simultaneously.
  3. Wash cells with ice cold PBS 2X by flooding the plate and decanting off wash
  4. Wash cells 1X with ice cold lysis buffer by flooding the plate and decanting off wash.
  5. Remove any remaining wash with a 1 ml pipette and add 1 ml of Lysis buffer containing 2X protease inhibitors to each plate.
  6. Scrape the cells off the plate and place in Dounce homogenizer. Incubate 5 minutes on ice.
  7. Rupture cells with 5-10 strokes of the homogenizer. Ruptured nuclei will release chromatin that will bind up the microsomes and reduce yield. It is better to under-homogenize than over. 70% lysis is a good target.
  8. Efficiency is checked by examination of lysate stained with Trypan blue using inverted microscope.
  9. Osmolarity is balanced by adding   5X sucrose buffer to homogenizer and mixing with 1 stroke
  10. Cell debris is pelleted at maximum  speed using clinical centrifuge at 4°C (3000 xg)
  11. Supernatant containing microsomes is spun at 16000  xg in refrigerated microfuge at 4°C for 30 minutes. Large volumes can be spun at 26000 rpm in ultracentrifuge using SW28 rotor
  12. Microsome pellet is resuspended in appropriate buffer for experiment.
  13. Bradford protein assay used to determine protein concentration.
  14. Samples are stored at -80°C

 

Lysis Buffer:

10 mM HEPES pH 7.2

1 mM EDTA

2x Protease Inhibitor cocktail

 

5X Sucrose Buffer:

1.25 M Sucrose

10 mM HEPES pH 7.2

 

1000X Protease Inhibitor Cocktail:

Leupeptin (Sigma L2884):    1mg/ml

Aprotinin (Sigma A1153):    2mg/ml

E64 (Sigma E3132):        3.57mg/ml

Benzamidine (Sigma B6506):    156.6mg/ml

Pefablock (Roche 1429876):    2M