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

Revision as of 20:37, 21 January 2011

1. Wash cells 3x in 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