Difference between revisions of "3T3-L1 Adipocyte Fractionation"
From Bridges Lab Protocols
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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== | ||
| Line 8: | Line 9: | ||
#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. | ||
Revision as of 12:38, 4 May 2009
Materials
- PBS
- 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
- 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 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 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 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.
- 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>
