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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...'
#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 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
#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
#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
#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