Difference between revisions of "Triglyceride Assay from Cells and Tissues"

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Revision as of 20:11, 30 March 2010

Materials

  • Homogenization Buffer (50 mM Tris, 5 mM EDTA, 30 mM Mannitol, PI inhibitor)
  • 10M KOH
  • Chloroform/Methanol Mixture (2:1)
  • Butanol Mixture: 3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol
  • Sigma Triglyceride Assay Kit (Cat 337-B)

Protocol

  1. Weigh out 200-500mg tissue (record weight for normalization)
  2. Homogenize with dounce homogenizer in 2 mL Homogenization Buffer.
  3. Remove 200 uL to a tube containing 5 uL KOH
  4. Mix by inverting
  5. Add 800 uL Chloroform/Methanol Mixture
  6. Vortex vigorously then sit at room temperature for 5 min
  7. Centrifuge 10min at 13 000 RPM
  8. Take 180 uL of the bottom layer into a fresh tube.
  9. Dry in fume hood overnight (or until completely dry)
  10. Add 50uL of Butanol Mixture
  11. Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample.