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

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==Protocol==
 
==Protocol==
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# Weigh out 30 mg tissue (record weight for normalization) on dry ice into round bottom eppendorf tube.  Add one stainless steel ball bearing.
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#Use 5 female or 8 Male flies for Assay
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# Add 500 uL Homogenization Buffer.
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#Homogenize flies (3 min @ 40Hz) in 1mL of .05% Tween (from Tween-20 stock)
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# Homogenize with qiagen tissue lyser 3 minutes at 25 Hz for Liver/WAT or 5 min at 30 Hz for muscle.
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#Heat samples in 70C waterbath for 5 minutes
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# Add 12.5 uL KOH
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#Spin samples @ 5000G for 1 minute
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# Mix by inverting
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#Transfer 500ul of supernatent in new microfuge tube
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# Add 800 uL '''Chloroform/Methanol Mixture'''
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#Spin @ 14000G for 3 minutes
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# Vortex vigorously then sit at room temperature for 5 min
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#Add 50ul of sample to 200ul of TG solution in a 96 well plate
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# Centrifuge 10min at 13 000 RPM
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#Incubate plate @ 37C for 5 mins
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# Take the bottom layer into a fresh labelled tube. 
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#Measure 540nm absorbance
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# Dry in fume hood overnight (or until completely dry)
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# If absorbance is going to be measured by cuvette, use non-bolded values.  If you are using a plate reader use bolded values.
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# Add 500uL '''(50uL)''' of '''Butanol Mixture'''. See [[#Suggested Volumes | Suggested Volumes]] for your specific tissue
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# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample:
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## Resuspend triglyceride and glycerol reagent with water if necessary.
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## Calculate how many samples you have (samples + buffer blank + 6 standard curve values).
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## 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.
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## Aliquot 700 uL into a cuvette or '''100 uL into a well of a 96 well plate'''.
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## For standards add 0-5 uL of glycerol standard.
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## Add 5 uL of resuspended lipid to each well to start (also make a 5uL blank of the butanol mixture) and mix.
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## 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.
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## Measure absorbance at 540 nm.
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## 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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Revision as of 21:11, 31 July 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. Use 5 female or 8 Male flies for Assay
  2. Homogenize flies (3 min @ 40Hz) in 1mL of .05% Tween (from Tween-20 stock)
  3. Heat samples in 70C waterbath for 5 minutes
  4. Spin samples @ 5000G for 1 minute
  5. Transfer 500ul of supernatent in new microfuge tube
  6. Spin @ 14000G for 3 minutes
  7. Add 50ul of sample to 200ul of TG solution in a 96 well plate
  8. Incubate plate @ 37C for 5 mins
  9. Measure 540nm absorbance
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