The DNA used in the polymerization reaction was fragmented by hydrodynamic shearing. The DNA was passed through the orifice of a 28-gauge hypodermic needle. This method of shearing generates 1.5-2.0 kb DNA fragments5. The polymerization reaction was performed in an uncoated 96-well microtiter plate. To the well was added: 2.5 μg histones (Trevigen/Upstate U.C. Biotechnology), 1 μg DNA (Sigma), 15 μM NAD + Bio-NAD (Trevigen) in a 17:1 or 1:1 ratio, 1 mM 3- aminobenzamide6 (inhibitor of PARP, Sigma) 0.5 U PARP enzyme (Trevigen) and PARP buffer (500 mM Tris-HCl, 250 mM MgCl2). The reaction was performed at room temperature, for 30 minutes. The plate was then washed 2 times using the DELFIA Platewash. Eu-labelled streptavidin was diluted in DELFIA Assay Buffer to 200 ng/ well and incubated for 30 minutes at room temperature. The plate was washed 4 times using the DELFIA Platewash, prior to addition of 200 μL/well DELFIA Enhancment Solution. The fluorescence was measured using VICTOR2 TM plate reader.
The data shows that the DELFIA timeresolved fluorescence assay for inhibition of PARP allows for the measurement of the incorporation of biotinylated NAD. This technology is therefore suitable for the screening of inhibitors of PARP where the formation of poly(ADP-ribose) chains is inhibited.
1. Satoh, M. S. and Lindahl, T. 1994. Role of poly(ADP-ribose) formation in DNA repair. Nature 356, 356-358
2. Lautier, D., Langeux, J., Thibodeau, L., Menard, L. and Poirier, G. G. 1993. Molecular and biochemical features of poly(ADP-ribose) metabolism. Mol Cell Biochem