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

Revision as of 17:10, 28 June 2013

Materials

• Homogenization Buffer (50 mM Tris pH 8, 5 mM EDTA, 30 mM Mannitol, PI inhibitor, can be made in bulk without the PI, PI added fresh)
• 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 TR0100)

Protocol

1. Weigh out 30 mg tissue (record weight for normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing.
2. Add 500 uL Homogenization Buffer.
3. Homogenize with qiagen tissue lyser 3 minutes at 25 Hz for Liver/WAT or 5 min at 30 Hz for muscle.
4. Add 12.5 uL KOH
5. Mix by inverting
6. Add 800 uL Chloroform/Methanol Mixture
7. Vortex vigorously then sit at room temperature for 5 min
8. Centrifuge 10min at 13 000 RPM
9. Take the bottom layer into a fresh labelled tube.
10. Dry in fume hood overnight (or until completely dry)
11. If absorbance is going to be measured by cuvette, use non-bolded values. If you are using a plate reader use bolded values.
12. Add 500uL (50uL) of Butanol Mixture. See Suggested Volumes for your specific tissue
13. Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample:
    1. Resuspend triglyceride and glycerol reagent with water if necessary.
    2. Calculate how many samples you have (samples + buffer blank + 6 standard curve values).
    3. Prepare reagent, you need 560 uL (80uL) of glycerol reagent and 140 uL (20uL) of triglyceride reagent. Make a bit extra and combine in a falcon tube.
    4. Aliquot 700 uL into a cuvette or 100 uL into a well of a 96 well plate.
    5. For standards add 0-5 uL of glycerol standard.
    6. Add 5 uL of resuspended lipid to each well to start (also make a 5uL blank of the butanol mixture) and mix.
    7. Let sit for ~30 min at room temperature (or 5 min at 37C if you are in a hurry). If using > 10 uL of butanol mixture the solution may be cloudy, let it settle and it should become more clear.
    8. Measure absorbance at 540 nm.
    9. If any samples are A540<0.1 or above the 5uL standard A540 then repeat with more or less lipid as required.