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β-oxidation assay

(Using 3H-palmitate)  


 The day before: Refresh media of cells to be analyzed.

 

The day of:

 

Preparation of reaction mixture:

  1. Make 22 mM unlabeled palmitic acid in pure ethanol:
  2. Palmitic acid is 256.4 g/mol, so 22 mM palmitic acid is 5.64 mg/mL, or, more practically, weigh out something close to 56.4 mg of it, and dissolve in 10 mL ethanol per 56.4 mg.
  3. Add 50 mCi (50 mL) of [9,10-3H]-palmitic acid to 30 mL of 22 mM palmitic acid.*
  4. Evaporate the liquid using the nitrogen gas blower thing in the fume hood, and resuspend in 300 mL* Krebs–Ringer buffer containing 10 mg/mL fatty-acid-free BSA:
  5. 94.7 mg Krebs­–Ringer powder (in refrigerator) in 10 mL ddH2O
  6. +100 mg fatty-acid-free BSA (in refrigerator)
  7. Incubate mixture at 37 C for 30 minutes.
  8. Add mixture to 5.7 mL* Krebs–Ringer buffer to dilute palmitic acid to a final concentration of 110 mM. This is the reaction mixture.

*I've found that the above volumes work per 4 plates of cells.


Labeling assay:

  1. Wash cells with PBS.
  2. Treat one plate from each group of cells to be the blank by adding 1.25 mL methanol to it for 1 minute. Aspirate methanol.
  3. Add 1.25 mL reaction mixture to each plate.
  4. Incubate 37 C for desired length of time.
  5. Pipet reaction mixtures into snap-cap falcon tubes.
  6. Wash with 2 mL PBS, pipet the PBS into the falcon tubes.
  7. Add AG 1-8X anion exchange resin to chromatography columns:
  8. In a beaker add several chunks of the resin to only a few mL of 1 N NaOH, such that the mixture is thick and slushy, not liquidy but still pipetable.
  9. Using a plastic squeeze-bulb pipette, add resin-NaOH to chromatography columns until the resin fills most of the narrow (bottom) portion of the columns. This leaves 4-5 mL of space on the top. You will need to add more resin-NaOH a few times to each column because the NaOH drips through, lowering the level of the resin from what you originally added. Make all of the columns have about the same amount of resin.
  10. Add 20 mL NaOH to the columns, a few mL at a time. Let it all drip though.
  11. Add the reaction mixtures to the columns and let drip into scintillation vials.
  12. Rinse columns with 2 x 1 mL ddH2O, letting ddH2O drip into vials.
  13. Add 10 mL scintillant to each scintillation vial.
  14. Lyse the cells remaining in your plates with an appropriate volume of Western lysis buffer and determine protein concentration of the lysates using the standard Bradford assay for proteins.

Using the Physiology department scintillation counter:

  1. Keyboard is on a sliding tray underneath the scintillation counter. Racks for vials are in the right-side drawers, flags for the racks are in the left-side drawers.
  2. Put your vials into one of the larger, black racks, as our vials don't fit into the gray ones.
  3. The flag you put into the left side of the scintillation rack is what tells the counter which program to run. Program 5 is best for 3H because it counts each vial for 5 minutes, which is better for the weakly radioactive 3H. Program 1 is also an option, but it only counts for 1 minute. *Make sure the little sliding tab on the flag is pushed out so that the scintillation counter will see it! If the tab is still pushed in, it will search for a flag without seeing one and never run a program!*
  4. With all your vials in the rack, invert them several times to mix, but not too vigorously. (Obviously hold another rack or something over the vials when you're inverting them so they don't fall out. That gravity's tricky.)
  5. When all the vials and flag number 5 are in the rack and the little tab in the flag is pushed out, put the rack in the back-right of the scintillation counter, close the lid, and push F2 on the keyboard to count the tritium.
  6. Repeat Program 5 with the vials in the reverse order, to make certain the readings of the last few vials weren�t skewed by settling of the scintillation mixture after waiting for too long. (This is probably pretty unlikely, but I do it.)

Troubleshooting the scintillation counter:

  • If nothing happens and the computer screen says 'Printer not ready': Push the on-line/off-line button on the printer.
  • If the scintillation counter moves the rack all over and it looks like it's doing something but nothing ever prints, it's probably because it's searching for a flag because you left the tab on the flag pushed in! Push it out!