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

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(updated with note about using less tissue)
(updated volumes and added a new section describing tissue specific volumes to use)
Line 14: Line 14:
 
# Vortex vigorously then sit at room temperature for 5 min
 
# Vortex vigorously then sit at room temperature for 5 min
 
# Centrifuge 10min at 13 000 RPM
 
# Centrifuge 10min at 13 000 RPM
−
# Take 180 uL of the bottom layer into a fresh tube.
+
# Take 200 uL of the bottom layer into a fresh tube. See [[#Suggested Volumes | Suggested Volumes]] for your specific tissue
 
# Dry in fume hood overnight (or until completely dry)
 
# Dry in fume hood overnight (or until completely dry)
 
# If absorbance is going to be measured by cuvette, use non-bolded values.  If you are using a plate reader use bolded values.
 
# If absorbance is going to be measured by cuvette, use non-bolded values.  If you are using a plate reader use bolded values.
−
# Add 50uL '''(500uL)''' of '''Butanol Mixture'''
+
# Add 50uL '''(500uL)''' of '''Butanol Mixture'''. See [[#Suggested Volumes | Suggested Volumes]] for your specific tissue
 
# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample:
 
# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample:
 
## Resuspend triglyceride and glycerol reagent with water if necessary.
 
## Resuspend triglyceride and glycerol reagent with water if necessary.
Line 25: Line 25:
 
## For standards add 0-5 uL of glycerol standard '''(or of a 1/10 dilution of the glycerol standard)'''.
 
## 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.
 
## 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).
+
## 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.
 
## Measure absorbance at 540 nm.
 
## 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.
 
## 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.
 +
{| border="1"
 +
|-
 +
! Tissue/Condition !! Lysis Volume !! Chloroform Volume !! Resuspension Volume !! Assay Volume
 +
|-
 +
| Liver || 1 mL || 200 uL || 500 uL || 5 uL
 +
|-
 +
| Muscle || 1 mL || 200 uL || 50 uL || 5 uL
 +
|}

Revision as of 14:15, 28 May 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). You can use less tissue if necessary by reducing the lysis volume (must be greater than 500 uL) or increasing the amount of chloroform layer removed (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.

Tissue/Condition Lysis Volume Chloroform Volume Resuspension Volume Assay Volume
Liver 1 mL 200 uL 500 uL 5 uL
Muscle 1 mL 200 uL 50 uL 5 uL
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