Based on Bakondi et al. J. Histochem Cytochem. 50(1):91-98, 2002
Principle :
Biotinylated NAD (bio-NAD) offers a non-radioactive alternative for the detection of PARP activity in cells and tissues. Bio-NAD has been shown by Zhang et al. to serve as a substrate for PARP (1). Bio-NAD has also been demonstrated to be suitable for the determination of PARP activation in a cellular ELISA system (2).
Treatment of cells with DNA breaking agents such as hydrogen peroxide or peroxynitrite or DNA alkylating compounds causes PARP activation. Following treatment with DNA damaging agents, digitonin-permeabilized cells are incubated with bio-NAD. Activated PARP cleaves bio-NAD into nicotinamide and biotinylated ADP-ribose and synthesizes (bio-ADP-ribose)n polymers covalently attached to nuclear proteins. The reaction product [(bio-ADP-ribose)n] is detected by streptavidin-peroxidase. Peroxidase reaction is developed with nickel-enhanced diaminobenzidine (DAB) substrate (alternatively, streptavidine-peroxidase + DAB can be substituted with straptavidine-fluorescence compound conjugates).
The method has been successfully adapted to detect PARP activation in tissues (3-4).
Material :
Digitonin solution
Dithiothreitol (DTT)
Biotinylated-NAD (Trevigen)
PJ34 (PARP inhibitor)
10 % TCA (trichloracetic acid)
30 % hydrogen peroxide
streptavidin-conjugated peroxidase
Xylene
Permanent mounting medium (e.g. Vectamount™ from Vector Laboratories, Burlingame, CA)
Solutions :
PBS pH 7,4
11.5 g Na2HPO4 (anhydrous)
2.62 g NaH2PO4 (1H2O)
9 g NaCl
Dissolve in 1 liter distilled water and adjust to pH 7.4
Reaction buffer (w/o DTT, digitonin and bio-NAD)
100 mM Tris, pH 8.0
10 mM MgCl2
To stain 10 coverslips in 24 well tissue culture plates (300 µl/well), prepare 3.5 ml of reaction buffer. Add digitonin solution (final concentration: 0.01 %), DTT (final concentration: 1mM) and 175 µl bio-NAD (Trevigen, final concentration: 12.5 µM) to 3.5 ml reaction buffer (2 µl/ml) and vortex. Prepare immediately before use.
Protocol description :
1) Grow cells on coverslips or chamber slides. Treat cells with putative PARP activating agents and place cells into a CO2 incubator. If using hydrogen peroxide or peroxynitrite for the treatment, incubate cells for 20-30 min (for other DNA damaging stimuli, optimal incubation time must be determined by the investigator).
2) Aspirate the culture medium from the cells and replace it with reaction buffer complemented with digitonin, DTT and bio-NAD (prepared immediately before use). To negative control samples, add PJ34 at a final concentration of 10 µM (or any other PARP inhibitor).
3) Place slides or coverslips into a 37°C incubator for 60 min (do not use a CO2 incubator at this step !).
4) Aspirate the reaction buffer from the cells and fix the cells in prechilled (-20°C) 95 % ethanol for 10 min in a -20°C freezer (if cells were grown in chamber slides, chambers can be removed from the slides after this step ; follow manufacturer`s instruction).
5) Following ethanol fixation, cover cells with prechilled (-20°C) 10 % TCA to inactivate poly(ADP-ribose) glycohydrolase. (10 min, -20°C).
6) Rinse slides or coverslips with PBS and block endogenous peroxidase activity by immersing them into 0.5% hydrogen peroxide for 15 min, at room temperature. (e.g. 1 ml 30 % hydrogen peroxide dissolved in 60 ml methanol).
7) Wash slides or coverslips with PBS (3 x 10 min) and then overlay cells with blocking reagent. Incubate for 30 min at room temperature.
8) Rinse coverslips with PBS and apply streptavidin-peroxidase (diluted 1:100 in PBS) for 30 min at room temperature.
9) Wash coverslips (4 x 5 min) with PBS and apply substrate solution for 10-15 min.
10) Wash coverslips with distilled water and dehydrate in increasing concentrations (70-95-100 %) of ethanol. Remove coverslips from tissue culture plate and rinse them with xylene (xylene dissolves plastic tissue culture plates !).
11) Mount coverslips on slides using a permanent mounting medium (e.g. Vectamount™).
Practical tips :
Care must be taken to prevent drying out of samples.
Cytospin preps can be overlaid with 100 µl reagents and covered with Parafilm™.
Coverslips (1 cm diameter) can be placed into 24 well tissue culture plates and covered with 300 µl reagents.
Cells grown in 8 well chamber slides (e.g. Nalge Nunc N° 154453) require about 300-400 µl reagents to cover the bottom of the wells.
References :
(1) Zhang J, Snyder SH. Purification of a nitric oxide-stimulated ADP-ribosylated protein using biotinylated beta-nicotinamide adenine dinucleotide. Biochemistry 32:2228-2233, 1993.
(2) Bakondi E, Bai P, Szabó E , Hunyadi J, Gergely P, Szabó C. and Virág L. Detection of poly(ADP-ribose) polymerase activation in oxidatively stressed cells and tissues using biotinylated NAD substrate. J. Histochem. Cytochem. 50(1):91-98, 2002.
(3) Szabó É, Virág L, Bakondi E, Gyüre L, Haskó G, Bai P, Hunyadi J, Gergely P, Szabó C Peroxynitrite production, DNA breakage and poly(ADP-ribose) polymerase activation in a mouse model of oxazolone-induced contact hypersensitivity. J. Invest. Dermatol. 117(1):74-80, 2001.
(4) Soriano FG, Virág L, Jagtap P, Szabó É, Mabley JG, , Liaudet L, Marton A, Hoyt DG, Murthy KGK, Salzman AL, Southan GJ, Szabó C: Diabetic endothelial dysfunction: the role of poly (ADP-ribose) polymerase activation. Nature Medicine, 7(1):108-113, 2001
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