Navigate this Article


Adipocyte Subcellular Fractionation   

How to cite Favorites Q&A Share your feedback Cited by

Materials and Reagents

  1. Fat tissue
  2. Bovine serum albumin (BSA)
  3. (-)-N6-(R-Phenyl-isopropyl)-adenosine
  4. Adenosine
  5. Sodium orthovanadate
  6. Ammonium bicarbonate
  7. Sucrose
  8. Tizma base
  9. Magnesium sulphate (MgSO4·7H2O)
  10. Potassium chloride (KCl)
  11. Sodium phosphate
  12. Sodium pyrophosphate
  13.  Monopotassium phosphate
  14. Iodoacetate
  15. EDTA
  16. EGTA
  17. Sodium fluoride
  18. Glucose
  19. Calcium Chloride
  20. HEPES (ICN Biomedicals)
  21. Glucose
  22. Protease inhibitors (Roche Diagnostics)
  23. NaCl 0,9% solution (see Recipes)
  24. Collagenase solution (see Recipes)
  25. KRHLP solution (see Recipes)
  26. KRHG solution (see Recipes)
  27. PES homogenization buffer (see Recipes)
  28. Tris-EDTA solution (see Recipes)
  29. Stock solutions (see Recipes)


  1. Beckman centrifuges and ultracentrifuges (Beckman Coulter)
  2. Rotors: JA-21, SW-41, TLS-55, and TLA-100 (Beckman Coulter)
  3. Probe sonicator
  4. Teflon/glas homogenizer (Thermo Fisher Scientific)
  5. Gauze bandage
  6. Beckman UltraClear tubes (Nalgene & Beckman Coulter)


  1. The fat tissue is rinsed with isotonic NaCl solution (0.9%) immediately after harvesting.
  2. The tissue is cut to small pieces and incubated with collagenase (1 ml g-1 fat) for 1 h at 37 °C.
  3. KRHLP-1% BSA buffer is added and the cells are filtered first through a single and then a double layer of gauze bandage.
  4. The cells are washed with KRHLP-1% BSA buffer until the lower phase is clear.
  5. The cells are diluted with the same buffer up to 10-15% and preincubated with PIA (1μl ml-1 cell suspension) and adenosine (0.5 μl ml-1) for 10-15 min at 37 °C. The buffer is changed to PES buffer (homogenization buffer) containing 2 mM sodium orthovanadate and protease inhibitors. Up to 20-30% cells.
  6. The cells are homogenized at RT with 5 strokes in a Teflon/glass homogenizer.
  7. The homogenate is transferred to Nalgene tubes and centrifuged for 20 min at 4 °C (JA-21, 14,000 rpm).
    From now on all steps are done at 4 °C. With cold spatula the fat on the top of the tubes is removed.
    The supernatant contains intracellular membrane vesicles (microsome fraction) and soluble proteins (cytosol fraction).
    The pellet contains in addition to the plasma membrane, mitochondria and nuclei.
  8. The supernatant is transferred to Beckman UltraClear tubes and centrifuged for 75 min (SW-41, 35,000).
  9. The super containing cytosolic proteins is transferred to 15-ml tubes and frozen. The pellet (microsome) is resuspended in 200 μl Tris-EDTA/2 mM sodium orthovanadate.
  10. The pellet is resuspended in 200 μl Tris-EDTA/2 mM sodium orthovanadate and placed carefully on 1.12 M sucrose solution (400 μl). Rinse the pellet tube with 400 μl Tris-EDTA/2 mM sodium orthovanadate and put on the sucrose solution. (600 μl of suspension can be loaded on the 400 μl of 1.12 M sucrose).
  11. The sucrose tube is centrifuged for 60 min (TLS-55, 46,000 rpm). The plasma membrane will stay at the top of the sucrose solution while the mitochondria and the nuclei will sediment.
  12. The plasma membrane band (400-600 μl) is transferred to a new tube and diluted up to 1,000 μl with Tris-EDTA/2 mM sodium orthovanadate. Vortex! 10% is saved as plasma membrane sample and the rest is for caveolae preparation.
  13. The plasma membrane fraction and the caveolae fraction are pelleted (20 min, TLA-100, 69,000 rpm). The plasma membrane sample is resuspended in 200 μl Tris-EDTA/2 mM vanadate and frozen.
  14. Caveolae sample is resuspended in 200 μl carbonate buffer (pH 11) (or 50 mM NH4HCO3 + 2 mM sodium orthovanadate) and transferred to sonication tube. Caveolae tube is rinsed with additional 200 ml and the volume in the sonication tube is adjusted to 2,000 ml.
  15. The sonication probe is cooled before and sonication is performed at 16 micron (3x 20 sec, with 60 sec intervals).
  16. The sonicated sample is diluted with 2 ml 90% sucrose and placed at the bottom of an ultracentrifuge tube containing 5-35% discontinuous sucrose gradient.
  17. The tube is centrifuged for 16-20 h (SW-41, 39,000).
  18. A light-scattering band (caveolae-enriched fraction) confide to the 5-35% sucrose interface is collected (1 ml) and diluted in Tris-EDTA + 2 mM sodium orthovanadate up to 4 ml.
  19. Caveolae are pelleted for 20 min (TLA-100, 69,000 rpm).


  1. NaCl 0,9% solution
    9 g NaCl in 1,000 ml H2O
  2. Collagenase solution (pH 7.5)

  3. KRHLP solution (pH 7.5)

  4. KRHG solution (pH 7.5)

  5. PES homogenization buffer (pH 7.4)

  6. Tris-EDTA solution (pH 7.4)

  7. Stock solutions
    1. KRH/KRHLP

    2. HEPES
      37.23 g HEPES
      900 ml H2O
      pH 7.3 (NaOH)
      Add water to 1,000 ml
    3. Glucose - 139 mM
      2.5 g. glucose in 100 ml H2O
    4. Adenosine - 100 μM
      13.4 mg adenosine in 500 ml H2O
    5. PIA - 100 μM (-)-N6-(R-Phenyl-isopropyl)-adenosine (adenosine analog)
      1 mM stock: 1.93 mg in 5 ml EtOH abs
      1µM stock: 1 ml 1 mM dilute in 9 ml H2O (total volume is 10 ml)
    6. Sucrose 1.12 M
      3.83 g sucrose in Tris-EDTA to total volume of 10 ml.


  1. Aboulaich, N., Vainonen, J. P., Stralfors, P. and Vener, A. V. (2004). Vectorial proteomics reveal targeting, phosphorylation and specific fragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes. Biochem J 383(Pt 2): 237-248.
  2. Gustavsson, J., Parpal, S., Karlsson, M., Ramsing, C., Thorn, H., Borg, M., Lindroth, M., Peterson, K. H., Magnusson, K. E. and Stralfors, P. (1999). Localization of the insulin receptor in caveolae of adipocyte plasma membrane. FASEB J 13(14): 1961-1971.
Please login or register for free to view full text
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Aboulaich, N. (2011). Adipocyte Subcellular Fractionation. Bio-101: e36. DOI: 10.21769/BioProtoc.36.

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.

You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.