Difference between revisions of "3T3-L1 Plasma Membrane Isolation"

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#Wash cells 3x in HES (20 mM Hepes, pH 7.4; 1 mM EDTA, 255 mM Sucrose)
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#Wash cells 3x in cold HES (20 mM Hepes, pH 7.4; 1 mM EDTA, 255 mM Sucrose)
 
#Dry cells well and add 0.6 mL HES (w/Protease Inhibitor) per 150 mm dish
 
#Dry cells well and add 0.6 mL HES (w/Protease Inhibitor) per 150 mm dish
 
#Scrape cells and homogenize with dounce homogenizer for 20 strokes
 
#Scrape cells and homogenize with dounce homogenizer for 20 strokes

Revision as of 20:38, 21 January 2011

  1. Wash cells 3x in cold HES (20 mM Hepes, pH 7.4; 1 mM EDTA, 255 mM Sucrose)
  2. Dry cells well and add 0.6 mL HES (w/Protease Inhibitor) per 150 mm dish
  3. Scrape cells and homogenize with dounce homogenizer for 20 strokes
  4. Centrifuge @ 3,000 rpm (800g) at 4 degrees for 10 minutes
  5. Collect supernatant and spin @ 3,000 rpm at 4 degrees for 5 minutes
  6. Collect supernatant and spin in TLA 100.3 rotor at 19,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
  7. Wash pellet in 1.0 mL HES and spin in TLA 100.3 rotor at 19,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
  8. Resuspend pellet in 1.0 mL HES and homogenize with dounce for 10 strokes
  9. Layer the resuspended pellet on top of 10 mL 40% sucrose/HES in a 13.5 mL ultracentrifuge tube (tube = Beckman #331372)
  10. Top off centrifuge tube with HES until nearly full
  11. Spin in ultracentrifuge with rotor SW41 (saltiel login = 5238) at 39,000 rpm at 4 degrees for 1 hour
  12. PM should appear as band at the interface of the HES and Sucrose fractions
  13. Remove HES buffer above PM fraction until close to PM fraction
  14. Collect ~ 0.8 mL PM containing fraction
  15. Add 2.6 mL of HES to dilute sucrose
  16. Spin in ultracentrifuge with TLA 100.3 rotor at 35,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
  17. Discard supernatent
  18. Pellet = purified plasma membrane