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

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==Protocol==
 
==Protocol==
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#Use 5 female or 8 Male flies for Assay
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#Weigh out 30mg tissue (record weights for normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing.
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#Homogenize flies (3 min @ 40Hz) in 1mL of .05% Tween (from Tween-20 stock)
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#Add 500ul Homogenization Buffer
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#Heat samples in 70C waterbath for 5 minutes
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#Homogenize with Qiagen Tissue Lyser for 3 minutes @ 25Hz for Liver/WAT or for 5 minutes @ 30Hz for muscle
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#Spin samples @ 5000G for 1 minute
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#Add 12.5ul KOH
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#Transfer 500ul of supernatent in new microfuge tube
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#Mix by inverting
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#Spin @ 14000G for 3 minutes
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#Add 800ul '''Chloroform/Methanol Mixture'''
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#Add 50ul of sample to 200ul of TG solution in a 96 well plate
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#Vortex vigorously then sit at room temperature for 5 minutes
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#Incubate plate @ 37C for 5 mins
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#Centrifuge for 10 minutes @ 13000G
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#Measure 540nm absorbance
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#Transfer the bottom layer into a new tube
 +
#Let evaporate overnight at room temperature
 +
#If absorbance is going to be measured by cuvette, use non-bolded values. If using a plate reader, used bolded values.
 +
#Add 500ul '''(50ul)''' of '''Butanol Mixture'''. See Suggested Volumes for your specific tissue.
 +
#Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample.
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##Resuspend triglyceride and glycerol reagent with water if necessary
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##Calculate how many sample you have (samples + blank  + standard curve)
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##Prepare reagent. You need 560ul '''(80ul)''' of glycerol reagent and 140ul '''(20ul)''' of triglyceride reagent. Make extra and combine in a Falcon tube.
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##Aliquot 700ul into a cuvette or '''100ul into a well of a 96 well plate'''
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##For standars, add 0-5ul of glycerol standard
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##Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix.
 +
##Let sit for ~30 mins @ room temperature (or 5mins @ 37C if you are in a hurry). If using >10ul of butanol mix the solution may be cloudy. Let it settle and it should become more clear.
 +
##Measure absorbance @ 540nm
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##If any samples are A540<0.1 or above the 5ul standard the A540, then repeat with more or less lipid as required.

Revision as of 21:26, 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. Weigh out 30mg tissue (record weights for normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing.
  2. Add 500ul Homogenization Buffer
  3. Homogenize with Qiagen Tissue Lyser for 3 minutes @ 25Hz for Liver/WAT or for 5 minutes @ 30Hz for muscle
  4. Add 12.5ul KOH
  5. Mix by inverting
  6. Add 800ul Chloroform/Methanol Mixture
  7. Vortex vigorously then sit at room temperature for 5 minutes
  8. Centrifuge for 10 minutes @ 13000G
  9. Transfer the bottom layer into a new tube
  10. Let evaporate overnight at room temperature
  11. If absorbance is going to be measured by cuvette, use non-bolded values. If using a plate reader, used 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 5ul of sample.
    1. Resuspend triglyceride and glycerol reagent with water if necessary
    2. Calculate how many sample you have (samples + blank + standard curve)
    3. Prepare reagent. You need 560ul (80ul) of glycerol reagent and 140ul (20ul) of triglyceride reagent. Make extra and combine in a Falcon tube.
    4. Aliquot 700ul into a cuvette or 100ul into a well of a 96 well plate
    5. For standars, add 0-5ul of glycerol standard
    6. Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix.
    7. Let sit for ~30 mins @ room temperature (or 5mins @ 37C if you are in a hurry). If using >10ul of butanol mix the solution may be cloudy. Let it settle and it should become more clear.
    8. Measure absorbance @ 540nm
    9. If any samples are A540<0.1 or above the 5ul standard the A540, then repeat with more or less lipid as required.
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