Triglyceride Assay from Cells and Tissues: Difference between revisions

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==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)

* '''Butanol Mixture''': 3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol

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#Vortex vigorously then sit at room temperature for 5 minutes

#Centrifuge for 10 minutes @ 13000G

#Transfer ~~400~~ ul of the bottom layer into a new tube

#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.

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##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.

##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

@@ Line 1: / Line 1: @@
 ==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)
 * '''Butanol Mixture''': 3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol
@@ Line 15: / Line 15: @@
 #Vortex vigorously then sit at room temperature for 5 minutes
 #Centrifuge for 10 minutes @ 13000G
-#Transfer 400 ul of the bottom layer into a new tube
+#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.
@@ Line 21: / Line 23: @@
 ##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.
+##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