Triglyceride Assay from Cells and Tissues: Difference between revisions
updated with note about using less tissue |
updated volumes and added a new section describing tissue specific volumes to use |
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# 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 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. | ||
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## 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. | |||
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! Tissue/Condition !! Lysis Volume !! Chloroform Volume !! Resuspension Volume !! Assay Volume | |||
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| Liver || 1 mL || 200 uL || 500 uL || 5 uL | |||
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| Muscle || 1 mL || 200 uL || 50 uL || 5 uL | |||
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