3T3-L1 Adipocyte Fractionation: Difference between revisions
Created page with '==Materials== *PBS *Lysis Buffer (25 mM HEPES, 250 mM Sucrose, PI tablet) *Optiprep ==Fractionation== #Wash cells twice with ice cold PBS -/- #Scrape 150 mm dish into 4 mL of Ly...' |
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*Lysis Buffer (25 mM HEPES, 250 mM Sucrose, PI tablet) | *Lysis Buffer (25 mM HEPES, 250 mM Sucrose, PI tablet) | ||
*Optiprep | *Optiprep | ||
*Cool centrifuges and rotors for JA17/JA25.5, TLA100.3 and NVT90 to 4C | |||
==Fractionation== | ==Fractionation== | ||
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#Scrape 150 mm dish into 4 mL of Lysis Buffer | #Scrape 150 mm dish into 4 mL of Lysis Buffer | ||
#Homogenize in Dounce Homogenizer 20X on ice | #Homogenize in Dounce Homogenizer 20X on ice | ||
#Centrifuge 5 min at 3000g and collect post-nuclear supernatant (PNS). If desired resuspend pellet in 2 mL as mitochondria/nuclear fraction (MN) | #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 4C. Resuspend pellet in 2 mL as plasma membrane (PM). Save sample. | #Centrifuge supernatant 15 min at 17 200g at 4C in 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 4C. Resuspend pellet in 2 mL as high density microsomes (HDM). Save sample | #Centrifuge supernatant 30 min at 48 000g at 4C in 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. | #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. | #To load equal volumes on a gel, load twice as much volume of PNS and cytosol as MN/PM/HDM/LDM. | ||