Difference between revisions of "3T3-L1 Adipocyte Fractionation"
From Bridges Lab Protocols
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==Materials== | ==Materials== | ||
*PBS | *PBS | ||
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#Homogenize in Dounce Homogenizer 20X on ice | #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 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 15 min at 17 200g at 4C in JA17 or JA25.5 with brake off. Resuspend pellet in 4 mL (2 mL if doing optiprep)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 30 min at 48 000g at 4C in JA17 or JA25.5 with brake off. Resuspend pellet in 4 mL (2 mL if doing optiprep)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 equal volumes of fractions. If doing optiprep, load twice as much volume of PNS and cytosol as MN/PM/HDM/LDM. |
| + | #PM purification: Dissolve PM pellet in 1 mL HES, dounce 10 times, then load to 2 mL of 40% sucrose-cusion. Spin with SW41 rotor 42.1K (100,000g) for 1 hour. Discard top 1 mL (fat) and harvest 0.8 mL of PM containing fraction. Add 2.6 mL of HES in the fraction to dilute sucrose. Spin at 35,000 rpm for 20 min to pellet down PM. | ||
==Optiprep Gradient== | ==Optiprep Gradient== | ||
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#Centrifuge 4h at 62 000 RPM in NVT90 at 4C. | #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. | #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 '''do not boil''' | + | #Store samples at -80. Make up SDS samples and '''do not boil if probing for GLUT4''' |
==References== | ==References== | ||
| − | + | PMID 17765682 | |
Latest revision as of 15:52, 18 January 2011
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 4 mL (2 mL if doing optiprep)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 4 mL (2 mL if doing optiprep)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 equal volumes of fractions. If doing optiprep, load twice as much volume of PNS and cytosol as MN/PM/HDM/LDM.
- PM purification: Dissolve PM pellet in 1 mL HES, dounce 10 times, then load to 2 mL of 40% sucrose-cusion. Spin with SW41 rotor 42.1K (100,000g) for 1 hour. Discard top 1 mL (fat) and harvest 0.8 mL of PM containing fraction. Add 2.6 mL of HES in the fraction to dilute sucrose. Spin at 35,000 rpm for 20 min to pellet down PM.
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 do not boil if probing for GLUT4
