Difference between revisions of "Triglyceride Assay from Cells and Tissues"
From Bridges Lab Protocols
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==Protocol== | ==Protocol== | ||
| − | # Weigh out | + | # Weigh out 30 mg tissue (record weight for normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing. |
| − | # Homogenize with | + | # Add 500 uL Homogenization Buffer. |
| − | # | + | # Homogenize with qiagen tissue lyser 3 minutes at 25 Hz for Liver/WAT or 5 min at 30 Hz for muscle. |
| + | # Add 12.5 uL KOH | ||
# Mix by inverting | # Mix by inverting | ||
# Add 800 uL '''Chloroform/Methanol Mixture''' | # Add 800 uL '''Chloroform/Methanol Mixture''' | ||
# 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 | + | # Take the bottom layer into a fresh labelled tube. |
# 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. | ||
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## 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. | ||
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Revision as of 15:24, 19 June 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
- Weigh out 30 mg tissue (record weight for normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing.
- Add 500 uL Homogenization Buffer.
- Homogenize with qiagen tissue lyser 3 minutes at 25 Hz for Liver/WAT or 5 min at 30 Hz for muscle.
- Add 12.5 uL KOH
- Mix by inverting
- Add 800 uL Chloroform/Methanol Mixture
- Vortex vigorously then sit at room temperature for 5 min
- Centrifuge 10min at 13 000 RPM
- Take the bottom layer into a fresh labelled tube.
- 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.
- Add 50uL (500uL) of Butanol Mixture. See Suggested Volumes for your specific tissue
- 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). 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.
- If any samples are A540<0.1 or above the 5uL standard A540 then repeat with more or less lipid as required.
