Difference between revisions of "Triglyceride Assay from Cells and Tissues"
From Bridges Lab Protocols
(updated volumes and lysis procedure) |
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==Materials== | ==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) | + | * '''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) (to make 50 mL of homogenization buffer - 2.5 mL of 1M Tris pH 8, 500 uL of 0.5 M EDTA, 0.273 g Mannitol, fill up the rest with water) |
| − | * 10M KOH | + | * 10M KOH (28.1g in 50 mL of water) |
* '''Chloroform/Methanol Mixture''' (2:1) | * '''Chloroform/Methanol Mixture''' (2:1) | ||
* '''Butanol Mixture''': 3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol | * '''Butanol Mixture''': 3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol | ||
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==Protocol== | ==Protocol== | ||
| − | # Weigh out 30 | + | #Weigh out 30-50mg tissue (record weights for normalization) on dry ice into round bottom eppendorf tube (2 mL). Add one stainless steel ball bearing. |
| − | # Add | + | #Add 500ul Homogenization Buffer |
| − | # Homogenize with | + | #Homogenize with Qiagen Tissue Lyser for 3 minutes @ 25Hz for Liver/WAT or for 5 minutes @ 30Hz for muscle |
| − | # Add 12. | + | #Add 12.5ul KOH |
| − | # Mix by inverting | + | #Mix by inverting then transfewr sample to a new 1.5 mL tube. Place dirty ball bearing in ethanol (located in the fume hood) |
| − | # Add | + | #Add 800ul '''Chloroform/Methanol Mixture''' |
| − | # Vortex vigorously then sit at room temperature for 5 | + | #Vortex vigorously then sit at room temperature for 5 minutes |
| − | # Centrifuge | + | #Centrifuge for 10 minutes @ 13000G |
| − | # | + | #Transfer 200 ul of the bottom layer into a new tube |
| − | # | + | #Centrifuge again for 7-10 minutes @ 13000G |
| − | # | + | #Transfer 150 ul of the bottom layer into the new tube (if there is enough of the bottom layer, transfer a total of 200 ul) |
| − | # Add | + | #Let evaporate overnight at room temperature |
| − | # Measure triglyceride levels using | + | #Add'''(50ul)''' of '''Butanol Mixture''' and vortex. See Suggested Volumes for your specific tissue. |
| − | ## Resuspend triglyceride and glycerol reagent with water if necessary | + | #Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample. |
| − | ## Calculate how many | + | ##Resuspend triglyceride and glycerol reagent with water if necessary |
| − | ## Prepare reagent | + | ##Calculate how many sample you have (samples + blank + standard curve) |
| − | ## Aliquot | + | ##Prepare reagent. You need 80uL of glycerol reagent and 20uL of triglyceride reagent. Make extra and combine in a Falcon tube. |
| − | ## For standards add 0-5 | + | ##Aliquot '''100ul into a well of a 96 well plate''' |
| − | ## Add | + | ##For standards, add 0-5 and .5ul of glycerol standard |
| − | ## Let sit for ~30 | + | ##Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix. |
| − | ## Measure absorbance | + | ##Pop any bubbles with tip before incubating. |
| − | ## If any samples are A540<0.1 or above the | + | ##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 | ||
| + | ##If any samples are A540<0.1 or above the 5ul standard the A540, then repeat with more or less lipid as required. | ||
Latest revision as of 17:03, 11 October 2019
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) (to make 50 mL of homogenization buffer - 2.5 mL of 1M Tris pH 8, 500 uL of 0.5 M EDTA, 0.273 g Mannitol, fill up the rest with water)
- 10M KOH (28.1g in 50 mL of water)
- 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 30-50mg tissue (record weights for normalization) on dry ice into round bottom eppendorf tube (2 mL). Add one stainless steel ball bearing.
- Add 500ul Homogenization Buffer
- Homogenize with Qiagen Tissue Lyser for 3 minutes @ 25Hz for Liver/WAT or for 5 minutes @ 30Hz for muscle
- Add 12.5ul KOH
- Mix by inverting then transfewr sample to a new 1.5 mL tube. Place dirty ball bearing in ethanol (located in the fume hood)
- Add 800ul Chloroform/Methanol Mixture
- Vortex vigorously then sit at room temperature for 5 minutes
- Centrifuge for 10 minutes @ 13000G
- Transfer 200 ul of the bottom layer into a new tube
- Centrifuge again for 7-10 minutes @ 13000G
- Transfer 150 ul of the bottom layer into the new tube (if there is enough of the bottom layer, transfer a total of 200 ul)
- Let evaporate overnight at room temperature
- Add(50ul) of Butanol Mixture and vortex. See Suggested Volumes for your specific tissue.
- Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample.
- Resuspend triglyceride and glycerol reagent with water if necessary
- Calculate how many sample you have (samples + blank + standard curve)
- Prepare reagent. You need 80uL of glycerol reagent and 20uL of triglyceride reagent. Make extra and combine in a Falcon tube.
- Aliquot 100ul into a well of a 96 well plate
- For standards, add 0-5 and .5ul of glycerol standard
- Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix.
- Pop any bubbles with tip before incubating.
- 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
- If any samples are A540<0.1 or above the 5ul standard the A540, then repeat with more or less lipid as required.
