19
edits
Changes
no edit summary
==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
==Protocol==
#Weigh out 30mg 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
#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 560ul '''(80ul)''' 80uL of glycerol reagent and 140ul '''(20ul)''' 20uL of triglyceride reagent. Make extra and combine in a Falcon tube.##Aliquot 700ul into a cuvette or '''100ul into a well of a 96 well plate'''##For standarsstandards, 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.
