3T3-L1 Plasma Membrane Isolation: Difference between revisions
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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 | ||
#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) | ||