|
|
| Line 7: |
Line 7: |
|
| |
|
| ==Protocol== | | ==Protocol== |
| # Weigh out 30 mg tissue (record weight for normalization) on dry ice into round bottom eppendorf tube. Add one stainless steel ball bearing. | | #Use 5 female or 8 Male flies for Assay |
| # Add 500 uL Homogenization Buffer.
| | #Homogenize flies (3 min @ 40Hz) in 1mL of .05% Tween (from Tween-20 stock) |
| # Homogenize with qiagen tissue lyser 3 minutes at 25 Hz for Liver/WAT or 5 min at 30 Hz for muscle. | | #Heat samples in 70C waterbath for 5 minutes |
| # Add 12.5 uL KOH | | #Spin samples @ 5000G for 1 minute |
| # Mix by inverting | | #Transfer 500ul of supernatent in new microfuge tube |
| # Add 800 uL '''Chloroform/Methanol Mixture'''
| | #Spin @ 14000G for 3 minutes |
| # Vortex vigorously then sit at room temperature for 5 min
| | #Add 50ul of sample to 200ul of TG solution in a 96 well plate |
| # Centrifuge 10min at 13 000 RPM | | #Incubate plate @ 37C for 5 mins |
| # Take the bottom layer into a fresh labelled tube.
| | #Measure 540nm absorbance |
| # Dry in fume hood overnight (or until completely dry) | |
| # If absorbance is going to be measured by cuvette, use non-bolded values. If you are using a plate reader use bolded values.
| |
| # Add 500uL '''(50uL)''' of '''Butanol Mixture'''. See [[#Suggested Volumes | Suggested Volumes]] for your specific tissue | |
| # Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample:
| |
| ## Resuspend triglyceride and glycerol reagent with water if necessary.
| |
| ## Calculate how many samples you have (samples + buffer blank + 6 standard curve values).
| |
| ## Prepare reagent, you need 560 uL '''(80uL)''' of glycerol reagent and 140 uL '''(20uL)''' of triglyceride reagent. Make a bit extra and combine in a falcon tube.
| |
| ## Aliquot 700 uL into a cuvette or '''100 uL into a well of a 96 well plate'''.
| |
| ## For standards add 0-5 uL of glycerol standard. | |
| ## Add 5 uL of resuspended lipid to each well to start (also make a 5uL blank of the butanol mixture) and mix.
| |
| ## Let sit for ~30 min at room temperature (or 5 min at 37C if you are in a hurry). If using > 10 uL of butanol mixture the solution may be cloudy, let it settle and it should become more clear.
| |
| ## Measure absorbance at 540 nm.
| |
| ## If any samples are A540<0.1 or above the 5uL standard A540 then repeat with more or less lipid as required.
| |