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

Revision as of 21:01, 11 July 2012

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 337-B)

Protocol

1. Weigh out 200-500mg tissue (record weight for normalization). You can use as low as 30 mg tissue if necessary by reducing the lysis volume (must be at least 500 uL) or increasing the amount of the lower (chloroform) layer removed up to 500 ul(normally 180 uL).
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 200 uL of the bottom layer into a fresh tube. See Suggested Volumes for your specific tissue
9. Dry in fume hood overnight (or until completely dry)
10. If absorbance is going to be measured by cuvette, use non-bolded values. If you are using a plate reader use bolded values.
11. Add 50uL (500uL) of Butanol Mixture. See Suggested Volumes for your specific tissue
12. 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 (or of a 1/10 dilution of the 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.

Suggested Volumes

This is based on using a 96 well plate to measure final concentrations.