Buffers:
- 2X buffer
10 ml 0.5 M imidazol, pH 6.6
0.21 g LiCl
1.25 ml 1 M MgCl2
1.0 ml 0.1 M EGTA, pH 6.6
--> Bring volume up to 50 ml with distilled water.
- dilution buffer
1 ml 0.5 M imidazol, pH 6.6
2.5 ml 20 mM diethylenetriaminepentaacetic acid (DTPA)
--> Bring volume up to 50 ml with distilled water.
Reaction Mixture:
solution [stock] vol/tube # samples total vol.
2X buffer - 50 µl X _______ = _______
DTT 1 M 0.2 µl X _______ = _______
DGK membrane 3.8 µg/µl 0.44 µl X _______ = _______
dilution buffer - 19.36 µl X _______ = _______
total = _______
1) Treat and harvest cells.
2) Extract lipids via a basic Bligh-Dyer extraction:
a) Resuspend cell pellet in 3 ml chloroform/methanol (1:2) & vortex.
b) Add 0.8 ml water & vortex.
c) Pellet cellular debris by spinning at 3,000 rpm (2000xg) for 5 min.
d) Add 1 ml chloroform & vortex.
e) Add 1 ml water & vortex.
f) Discard upper, aqueous phase.
3) Collect 1.2-1.5 ml of the lower phase (total volume of lower phase should constitute 2 ml, but to prevent any aqueous contamination only a portion is used).
---> Also aliquot standards: 10, 20, 40, 80 & 160 pmoles of ceramide and diolein.
4) Dry down lipids using extra dry nitrogen gas.
5) Resuspend lipids in each tube in 20 µl of b-OG/DOPG (7.5%:25 mM mixed micelle) and vortex vigorously.
---> Have a blank tube containing b-OG/DOPG only.
6) Sonicate samples briefly and again vortex vigorously.
---> This step is only necessary if the lipid is not readily "going into solution."
7) Add 70 µl of reaction mixture to each sample and vortex vigorously.
8) Add 10 µl of 2 mM, 4 µCi [g-32P] ATP to each tube to initiate reaction.
---> When preparing the mixture of hot and cold ATP assume that the hot ATP constitutes insignificant concentrations of ATP.
9) Allow reaction to proceed for 30 minutes at room temperature.
10) Stop reaction with 3 ml of chloroform/methanol (1:2) & vortex.
11) Add 0.7 ml water to each sample & vortex.
---> Some protocols use 1% HClO4 instead of water at this step and at step 13.
12) Add 1 ml of chloroform to each sample & vortex.
13) Add 1 ml water to each sample & vortex.
14) Spin samples @ 3,000 rpm (2000xg) for 5 minutes.
15) Aspirate off the upper phase and collect 1.2-1.5 ml of lower phase.
NOTE: The upper phase contains most of the radioactivity, so be very careful when aspirating!
16) Dry down samples, resuspend in chloroform and spot on TLC plates (Whatman Silica Gel 60A plates, catalog #: 4866-821).
17) Develop plates in TLC chamber containing the following solvent system:
---> chloroform/acetone/methanol/acetic acid/water
(10:4:3:2:1)
18) Expose film overnight.
19) Develop film, scrape spots & count ceramide phosphate, phosphatidic acid and lyso-phosphatidic acid spots.
