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

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(wrote initial page)
 
(added details about using the sigma kit)
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# Take 180 uL of the bottom layer into a fresh tube.
 
# Take 180 uL of the bottom layer into a fresh tube.
 
# Dry in fume hood overnight (or until completely dry)
 
# Dry in fume hood overnight (or until completely dry)
−
# Add 50uL of '''Butanol Mixture'''
+
# If absorbance is going to be measured by cuvette, use non-bolded values.  If you are using a plate reader use bolded values.
−
# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample.
+
# Add 50uL '''(500uL)''' of '''Butanol Mixture'''
 +
# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample:
 +
## Resuspend triglyceride and glycerol reagent with water if necessary.
 +
## Calculate how many samples you have (samples + buffer blank + 6 standard curve values).
 +
## 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.
 +
## Aliquot 700 uL into a cuvette or '''100 uL into a well of a 96 well plate'''.
 +
## For standards add 0-5 uL of glycerol standard '''(or of a 1/10 dilution of the glycerol standard)'''.
 +
## Add 5 uL of resuspended lipid to each well to start (also make a 5uL blank of the butanol mixture) and mix.
 +
## Let sit for ~30 min at room temperature (or 5 min at 37C if you are in a hurry).
 +
## Measure absorbance at 540 nm.
 +
## If any samples are A540<0.1 or above the 5uL standard A540 then repeat with more or less lipid as required.

Revision as of 16:03, 13 April 2012

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. 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
  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).
    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.
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