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

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.