Solutions :
NAD 1 µM
HClO4 (PCA) 0.5 M
KOH 1M/K2HPO4-KH2PO4 0.33 M pH 7.5
PCA/KOH 1M/K2HPO4-KH2PO4 0.33 M pH 7.5 (2:1)
Phenazine ethosulfate (PES) 20 mM (store at 4°C and protect from light)
EDTA 500 mM pH 8
MTT tetrazolium 5 mM (store at 4°C and protect from light)
Iodoacetate 12 mM (store at 4°C)
Bicine 100 mM pH 7.8
Bicine 1.2 M pH 7.8
BSA 10 mg/ml
EtOH 6 M (34.8%)
Alcohol deshydrogenase (220 U/mg): 10 mg/ml stock in H20 (store at -20°C). Dilute at 0.5 mg/ml (1x) in bicine
Reaction buffer: 310 µl
- 60 µl Bicine 1.2M pH7.8
- 60 µl BSA 10 mg/ml
- 60 µl EtOH 6 M
- 6 µl EDTA 500 mM
- 60 µl PES 20 mM
- 60 µl MTT 5 mM
- 4 µl H2O
Sample preparation :
1. Harvest 2-4.105 cells / 6 cm dish in triplicate
2. At T = 0, treat cells for t min with DNA damaging agent
3. At T = t, wash quickly with ice cold PBS ; carefully remove all the PBS
4. Add 1 ml cold PCA 0.5 M ; incubate 20 min on ice
5. Take 900 µl and add into 450 µl KOH 1M/K2HPO4-KH2PO4 0.33 M pH 7.5 ; vortex
6. Incubate 20 min on ice or store at -20°C
7. Before use, centrifuge 2 min at 10,000 rpm, 4°C and keep the supernatant on ice
Reaction :
1. To 310 µl of reaction buffer, add 350 µl of sample ; incubate 5 min at 30°C
2. At T = 0, add 60 µl Alcohol dehydrogenase 1x and incubate 30 min at 30°C
3. At T = 30 min, add 300 µl iodoacetate 12 mM
4. Read O.D. at 570 nm
Notes :
Make a standard curve with [NAD] ranging from 0-180 µM diluted in a solution PCA:KOH/Phosphate (2:1)
If the sample is too concentrated, dilute with PCA:KOH/Phosphate (2:1)
Reference :
Jacobson E. and Jacobson M.K. (1976) Arch. Biochem. Biophys. 175, 627-634.
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