9 May 2003

Reagents:

  • 14C Acetic acid sodium salt, Amersham CFA.13, 0.2 µCi/µl

  • 1x PBS

  • H2O

  • Chloroform, Methanol, Hexane, Isopropanol, 4 L, Stores

  • Long glass tubes and glass pipettes

  • Micropipette, VWR 53432-783

  • TLC Silica gel 60 (20 x 20 cm), VWR EM-11845-7, 25/$231.53

  • Carrier lipid stocks; UC (unstearified cholesterol) 1 mg/ml, CE (cholesterol ester) 0.5 mg/ml, TG (triglyceride) 1 mg/ml.

  • Neutrolipid solvent (N.L.); 108 ml hexethane, 1.2 ml HAc (glacial), 30 ml Diethyl Ether (anhydrous); available at Stores

  • Iodine Crystals

  • Scintillation Fluid (Ecolume) and vials (Wheaton #986644)

 

Protocol:

  1. Set up cultures at 5 x 105 cells/ml in 2 ml with 14C acetate (10 µl at 0.2 µCi/µl --> final 1 µCi/µl).

  2. Grow 1 to 2 hours.

  3. Wash with 1x PBS (important to remove FBS).

  4. Store cell pellet at -20°C until ready for further processing.

  5. Add 1 ml Hexane:Isopropanol 3:1 v/v (to extract lipids from pellet).

  6. Vortex.

  7. Incubate 30 minutes at RT.

  8. Transfer supernatant to new glass tube careful not to touch the pellet.

  9. Wash pellet with 1 ml Hexane:Isopropanol. Transfer supernatant to same glass tube.

  10. Evaporate to dryness under air stream in 40°C heat block.

  11. Add 4 ml Folch (chloroform:methanol 2:1 v/v).

  12. Add 1 ml H2O.

  13. Add 20 µg complete carrier lipid to each sample.

  14. Vortex.

  15. Incubate 20-30 minutes at RT.

  16. Vortex.

  17. Spin for 10 minutes at 2000 rpm.

  18. Aspirate top aqueous phase, keep organic phase.

  19. Add 1 ml H2O.

  20. Vortex.

  21. Spin for 10 minutes at 2000 rpm.

  22. Aspirate top aqueous phase, keep organic phase.

  23. Evaporate to dryness under air stream in 40°C heat block.

  24. Rinse sides with chloroform.

  25. Evaporate to dryness under air stream in 40°C heat block.

  26. Resuspend in 110 µl chloroform.

  27. Spot at ~1.5 cm from bottom of gel (above tank solvent).

    1. Spot carrier alone for a reference band.

  1. Place gel in tank with solvent and run.

    1. Run until gel is completely saturated ~20 min.

    2. Dry gel on 40°C heat block.

  1. Place gel in tank of fresh iodine crystals until all bands are clearly visible.

  2. Mark bands of interest with pencil.

  3. Dry gel until iodine stained bands are clearly not visible.

  4. Cut marked bands into scintillation vials and add 4.5 ml scintillation fluid.

Reference: Oram Lab, Medicine, Metabolism & Endocrinology, University of Washington

Department of Microbiology
School of Medicine
University of Washington
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