Material :
TCA (trichloracetic acid)
HEPES (Sigma-Aldrich ; H-1016 ; 100 g)
Digitonin (Sigma-Aldrich ; D-1407 ; 1 g)
[3H]-NAD+ (DuPont NEN ; NET-443 ; 50 µCi/250ml)
NAD+ (Boehringer-Mannheim ; cat. # 775 754)
Solutions :
Stock Buffer (100 ml)
56 mM HEPES pH 7.5 1.330 g
28 mM KCl 0.208 g
28 mM NaCl 0.164 g
2 mM MgCl2 0.041g
This stock can be stored for weeks at 4°C.
Add digitonin, cold and hot NAD fresh before use.
0.01% digitonin 10 mg/100 ml
0.125 µM (non-tritiated) NAD+ 0.829 mg/ml (dil. 1:10,000)
2% SDS/0.1 N NaOH solution
Protocol description :
1. For each 12-well plate, you will need 6.0 ml buffer, so take about 7 ml per plate and put them into a 50 ml conical tube ; thaw out the [3H]-NAD stock and add 5 µl/ml of buffer to reach a final concentration of 0.5 µCi/ml. Place the conical tube in a 37°C water bath.
2. When the cells are ready to be assayed, aspirate off the culture medium from all the wells, and immediately add 0.5 ml per well of tritiated NAD solution using an Eppendorf repeater. Put the plates in 37°C incubator for 10 min.
3. Take a 25 mm disposable cell scraper and scrape the cells in the wells. Transfer each well content to a labeled Eppendorf tube. Add 200 ml of ice-cold 50% TCA to each tube, cap the tubes and refrigerate them for ~ 3 hours.
4. After TCA precipitation, centrifuge the tubes at 10,000 rpm for 10 min.
5. Remove the supernatant from each tube and wash the pellet with 500 µl of ice-cold 5% TCA, centrifuge at 10,000 rpm for 5 min.
6. Repeat step 5 twice.
7. After removal of the last TCA wash, add in each tube 500 ml of a 2% SDS/0.1 N NaOH solution, cap tubes and solubilize overnight in a 37°C incubator.
8. Transfer the content of each tube into a counting vial with 7 ml of scintillation cocktail. Count 2 min in a beta scintillation counter (tritium window).
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