3T3-L1 Adipocyte Fractionation
From Bridges Lab Protocols
- Lysis Buffer (25 mM HEPES, 250 mM Sucrose, PI tablet)
- Cool centrifuges and rotors for JA17/JA25.5, TLA100.3 and NVT90 to 4C
- 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.
- 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