Welcome guest, Sign in

Home

X
加载中

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

[Bio101] Adipocyte Subcellular Fractionation

Cell Biology > Organelle isolation > Caveolae

[Abstract]

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)

Equipment

  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)

Procedure

  1. 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).

References

  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.


How to cite: Aboulaich, N. (2011). Adipocyte Subcellular Fractionation. Bio-protocol Bio101: e36. DOI: 10.21769/BioProtoc.36; Full Text



Share Your Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Q&A and Troubleshooting:

  • Add Photo
  • Add Video

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 (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.


Login | Register
How to cite
Share
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook