3T3-L1 Plasma Membrane Isolation: Difference between revisions
Created page with '#Wash cells 3x in 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...' |
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#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) | #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 | #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) | ||
#Top off centrifuge tube with HES until nearly full | #Top off centrifuge tube with HES until nearly full | ||