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*Lysis Buffer (25 mM HEPES, 250 mM Sucrose, PI tablet)
*Optiprep
*Cool centrifuges and rotors for JA17/JA25.5, TLA100.3 and NVT90 to 4C
==Fractionation==
#Scrape 150 mm dish into 4 mL of Lysis Buffer
#Homogenize in Dounce Homogenizer 20X on ice
#Centrifuge 5 min at 3000g in JA17 or JA25.5 and collect post-nuclear supernatant (PNS). If desired resuspend pellet in 2 mL as mitochondria/nuclear fraction (MN)#Centrifuge supernatant 15 min at 17 200g at 4Cin JA17 or JA25.5 with brake off. Resuspend pellet in 2 mL as plasma membrane (PM). Save sample.#Centrifuge supernatant 30 min at 48 000g at 4Cin JA17 or JA25.5 with brake off. Resuspend pellet in 2 mL as high density microsomes (HDM). Save sample
#Centrifuge supernatant 75 min at 90 000 RPM in TLA 100.3 at 4C. Pellet is low density microsomes (LDM), supernatant is cytosol save a sample.
#To load equal volumes on a gel, load twice as much volume of PNS and cytosol as MN/PM/HDM/LDM.