Difference between revisions of "3T3-L1 Plasma Membrane Isolation"
From Bridges Lab Protocols
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| − | #Wash cells 3x in HES (20 mM Hepes, pH 7.4; 1 mM EDTA, 255 mM Sucrose) | + | #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
- 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
- Scrape cells and homogenize with dounce homogenizer for 20 strokes
- Centrifuge @ 3,000 rpm (800g) at 4 degrees for 10 minutes
- Collect supernatant and spin @ 3,000 rpm at 4 degrees for 5 minutes
- Collect supernatant and spin in TLA 100.3 rotor at 19,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
- 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)
- Resuspend pellet in 1.0 mL HES and homogenize with dounce for 10 strokes
- Layer the resuspended pellet on top of 10 mL 40% sucrose/HES in a 13.5 mL ultracentrifuge tube (tube = Beckman #331372)
- Top off centrifuge tube with HES until nearly full
- Spin in ultracentrifuge with rotor SW41 (saltiel login = 5238) at 39,000 rpm at 4 degrees for 1 hour
- PM should appear as band at the interface of the HES and Sucrose fractions
- Remove HES buffer above PM fraction until close to PM fraction
- Collect ~ 0.8 mL PM containing fraction
- Add 2.6 mL of HES to dilute sucrose
- Spin in ultracentrifuge with TLA 100.3 rotor at 35,000 rpm for 20 minutes at 4 degress (Tube = Beckman #349622)
- Discard supernatent
- Pellet = purified plasma membrane
