Materials :
• Glass assay tube, 25 mm diameter Whatman Glass Microfibre Filters or equivalent, 25 mm diameter vacuum filtration unit
• Liquid scintillation counter, high flash-point LSC-cocktail for aqueous and non aqueous samples, Ultima Gold MV - Packard;
• 5% TCA, 1 % inorganic phosphate solution, 95% ethanol.
PARP activity reaction :
Incubation Mixture
Tris-HCl pH 8.0 . . . . . . . . . . . . . . . . . . . . .50 mM
MgCl2 . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 4 mM
DTT . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 µM
DNase I activated DNA . . . . . . . . . . . . . 200 ng
BSA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 µg/ µl
NAD . . . . . . . . . . . . . . . . . . . . .. . . . . . . 400 µM final
[32P]-NAD . . . . . . . . . . . . . . . . . . . . . . . . 0.1 µCi/reaction
PARP-1 or PARP-2 . . . . . . . . . . . . . . . ..200 ng
H2O for 100 µl final
Protocol description :
1. The reaction should be done in triplicates. In a glass tube, incubate the reaction mixture for 10 min at room temperature; stop the reaction by adding 4 ml of 5% TCA, 1 % inorganic phosphate (that will precipitate the protein and the polymer);
2. Filter the reaction solution with the vacuum filtering system (the reaction specific material, poly(ADP-ribose), is retained onto the filter); wash the glass tube 3 times with 4 ml of 5% TCA, 1 % inorganic phospate, then with 4 ml of 95% ethanol. Dry the filter and count the radioactivity in a liquid scintillation counter.
References :
1. Simonin F, Poch O, Delarue M, de Murcia G. Identification of potential activesite residues in the human poly(ADP-ribose) polymerase. J. Biol. Chem. 1993 Apr 25;268(12):8529-35.
2. Amé JC, Rolli V, Schreiber V, Niedergang C, Apiou F, Decker P, Muller S, Hoger T, Ménissier-de Murcia J, de Murcia G. PARP-2, A novel mammalian DNA damage-dependent poly(ADP-ribose) polymerase. J. Biol. Chem. 1999 Jun 18;274(25):17860-8.
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