Triglyceride Assay from Cells and Tissues: Difference between revisions

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 ==Protocol==
-#Use 5 female or 8 Male flies for Assay
+#Weigh out 30mg tissue (record weights for normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing.
-#Homogenize flies (3 min @ 40Hz) in 1mL of .05% Tween (from Tween-20 stock)
+#Add 500ul Homogenization Buffer
-#Heat samples in 70C waterbath for 5 minutes
+#Homogenize with Qiagen Tissue Lyser for 3 minutes @ 25Hz for Liver/WAT or for 5 minutes @ 30Hz for muscle
-#Spin samples @ 5000G for 1 minute
+#Add 12.5ul KOH
-#Transfer 500ul of supernatent in new microfuge tube
+#Mix by inverting
-#Spin @ 14000G for 3 minutes
+#Add 800ul '''Chloroform/Methanol Mixture'''
-#Add 50ul of sample to 200ul of TG solution in a 96 well plate
+#Vortex vigorously then sit at room temperature for 5 minutes
-#Incubate plate @ 37C for 5 mins
+#Centrifuge for 10 minutes @ 13000G
-#Measure 540nm absorbance
+#Transfer the bottom layer into a new tube
+#Let evaporate overnight at room temperature
+#If absorbance is going to be measured by cuvette, use non-bolded values. If using a plate reader, used bolded values.
+#Add 500ul '''(50ul)''' of '''Butanol Mixture'''. 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 560ul '''(80ul)''' of glycerol reagent and 140ul '''(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 standars, add 0-5ul of glycerol standard
+##Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix.
+##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.