AA&MetaboliteQuantitationofCellPelletsPostAALabeling

Extraction:

1) Following spin, save supernatant for analysis. Be extremely careful not to disturb the pellet since it is somewhat dispersed.

2) Immediately upon removing supernatant from each tube, add 3 mls of chloroform-methanol, 1:2, and vortex vigorously!!

--> It is necessary to add the C:M immediately in order to get good dispersion of the pellet. If too much time passes prior to resuspension, the pellet does not dissolve well and sonication is necessary.

3) Add 0.8 ml of water and vortex vigorously.

--> Still have a monophase and it may be best to allow this monophase to equilibrate for a little while.

--> This is a good point to stop by storing the samples in the -200C freezer.

4) Spin out all large debris by spinning at 3,000 rpm for 5 mins.

5) Transfer the supernatant to a new pyrex glass tube and vortex again. Do not transfer any of the pellet, even if it is necessary to sacrifice some of the sample.

6) Add 1 ml of chloroform to break phases and vortex vigorously.

7) Add 1 ml of water and again vortex vigorously.

8) Spin samples at 3,000 rpm for 5 mins. to completely separate phases.

9) Aspirate off the top phase and the interface.

--> Remove enough such that no aqueous material remains at the miniscus.

10) The lower phase should constitute ~2 ml. Transfer 1.5-1.7 ml of this to a new pyrex glass tube.

--> This is a good point to stop by storing the transferred samples in the -200C freezer.

11) Dry down samples completely and resuspend lipids in 60µl of chloroform.

12) Use 20µl to spot a linear K TLC plate for TLC system #1, use 20µl to spot a linear K TLC plate for TLC system #2.

TLC :

A) Allow TLC tanks to equilibrate 1-2 hrs with solvent prior to running.

SYSTEM #1

--> SeparatesPA, TXB2, HHT, HETE & AA. 
ethylacetate/2,2,4-trimethylpentane/acetic acid/ H2O at a ratio of 90:50:20:100

SYSTEM #2

--> Separates DAG & AA.
benzene/diethyl ether/ethanol/NH3 at a ratio of 100:80:4:0.2

B) Following TLC remove plates and allow to evaporate completely. This should take a few hours.

--> If another plate needs to be run in the same tank then allow tank to equilibrate another hour following removal of the first plate.

C) Spray plate with enhance, spot with radioactive ink, wrap in saran wrap and place in a cassette (no intensifying screen necessary).

--> Spot ink in locations where it will not be disturbed. In other words, do not put spots below the sample application levels, since the plate will be held upside down to scrape spots.

18) Expose film for a minimum of 40 hours (3 days is much better) in the -800C freezer.

19) Allow enhance to completely evaporate.

--> Since the plate is wrapped in saran wrap during developing, enhance remains wet on the plate, and this must be evaporated in order to retain all of the silica when scraping.

20) Following film development, mark off locations of spots.

21) Hold plate upside down and tilted over glassine weigh papers and scrape spots. 

--> Add scrapings into prefilled plastic scintillation vials to minimize loss of silica and sample.

22) Vortex vials and count spots, count vials again 24 hours later.