Triglyceride Assay from Cells and Tissues: Difference between revisions
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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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##Resuspend triglyceride and glycerol reagent with water if necessary | ##Resuspend triglyceride and glycerol reagent with water if necessary | ||
##Calculate how many sample you have (samples + blank + standard curve) | ##Calculate how many sample you have (samples + blank + standard curve) | ||
##Prepare reagent. You need | ##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''' | ##Aliquot '''100ul into a well of a 96 well plate''' | ||
##For standards, add 0-5 and .5ul of glycerol standard | ##For standards, add 0-5 and .5ul of glycerol standard | ||