Difference between revisions of "3T3-L1 Plasma Membrane Isolation"
From Bridges Lab Protocols
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#Scrape cells and homogenize with dounce homogenizer for 20 strokes | #Scrape cells and homogenize with dounce homogenizer for 20 strokes | ||
#Centrifuge @ 3,000 rpm (800g) at 4 degrees for 10 minutes | #Centrifuge @ 3,000 rpm (800g) at 4 degrees for 10 minutes | ||
| − | #Collect supernatant and spin @ 3,000 rpm at 4 degrees for 5 minutes | + | #(Optional) 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) | #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) | + | #(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) |
| + | #(Optional) To further purify PM, resuspend in 0.6 mL HES and repeat steps #4 and #6 | ||
#Resuspend pellet in 1.0 mL HES and homogenize with dounce for 10 strokes | #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) | #Layer the resuspended pellet on top of 10 mL 40% sucrose/HES in a 13.5 mL ultracentrifuge tube (tube = Beckman #331372) | ||
Latest revision as of 21:44, 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
- (Optional) 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)
- (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)
- (Optional) To further purify PM, resuspend in 0.6 mL HES and repeat steps #4 and #6
- 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
