Open main menu

3T3-L1 Plasma Membrane Isolation

Revision as of 21:44, 21 January 2011 by Buchnerd (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Revision as of 21:44, 21 January 2011 by Buchnerd (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
  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. (Optional) 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. (Optional) 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. (Optional) To further purify PM, resuspend in 0.6 mL HES and repeat steps #4 and #6
  9. Resuspend pellet in 1.0 mL HES and homogenize with dounce for 10 strokes
  10. Layer the resuspended pellet on top of 10 mL 40% sucrose/HES in a 13.5 mL ultracentrifuge tube (tube = Beckman #331372)
  11. Top off centrifuge tube with HES until nearly full
  12. Spin in ultracentrifuge with rotor SW41 (saltiel login = 5238) at 39,000 rpm at 4 degrees for 1 hour
  13. PM should appear as band at the interface of the HES and Sucrose fractions
  14. Remove HES buffer above PM fraction until close to PM fraction
  15. Collect ~ 0.8 mL PM containing fraction
  16. Add 2.6 mL of HES to dilute sucrose
  17. Spin in ultracentrifuge with TLA 100.3 rotor at 35,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
  18. Discard supernatent
  19. Pellet = purified plasma membrane
Last modified on 21 January 2011, at 21:44