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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...'
==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 Lysis Buffer
#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 supernatant 15 min at 17 200g at 4C. 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 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.
==Optiprep Gradient==
#Wash LDM fraction once with lysis buffer and resuspend in 2 mL of lysis buffer. Save sample as LDM
#Gently homogenize 10X in Dounce Homogenizer
#Mix optiprep to generate 18% gradient (1.5 mL optiprep + 3.5 mL LDM)
#Centrifuge 4h at 62 000 RPM in NVT90 at 4C.
#Cut off top of tube and gently remove 350 uL fractions to fresh tubes on ice.
#Store samples at -80. Make up SDS samples and <bold>do not boil</bold>
==References==
<pubmed>17765682</pubmed>
*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 Lysis Buffer
#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 supernatant 15 min at 17 200g at 4C. 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 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.
==Optiprep Gradient==
#Wash LDM fraction once with lysis buffer and resuspend in 2 mL of lysis buffer. Save sample as LDM
#Gently homogenize 10X in Dounce Homogenizer
#Mix optiprep to generate 18% gradient (1.5 mL optiprep + 3.5 mL LDM)
#Centrifuge 4h at 62 000 RPM in NVT90 at 4C.
#Cut off top of tube and gently remove 350 uL fractions to fresh tubes on ice.
#Store samples at -80. Make up SDS samples and <bold>do not boil</bold>
==References==
<pubmed>17765682</pubmed>
