A scintillation proximity assay for poly(ADP-ribose) polymerase.
Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear protein in most of the eukaryotic tissues. When activated by DNA damage, PARP synthesizes poly(ADP-ribose) from NAD. Conventional radioactive PARP enzyme assay requires the separation of the polymer product from the NAD substrate, a rate-limiting step that hampers large-scale chemical library screening to identify novel small-molecule PARP inhibitors. By using biotinylated NAD, we have developed a scintillation proximity assay (SPA) for PARP. We demonstrated that PARP can incorporate the biotinylated ADP-ribose units into the radioactive poly(ADP-ribose) polymer, which can directly bind and excite the streptavidin-conjugated scintillation beads. PARP-SPA can be readily adapted to a 96-well format for automatic high-throughput screening for PARP inhibitors.
Cheung
A
A
Guilford Pharmaceuticals Inc., 6611 Tributary Street, Baltimore, Maryland, 21224, USA.
Zhang
J
J
D000818:Animals; D001345:Autoradiography; D001549:Benzamides / Q000494:pharmacology; D019921:Biotinylation; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D020128:Inhibitory Concentration 50; D051379:Mice; D009243:NAD / Q000378:metabolism; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis* / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012588:Scintillation Counting / Q000379:methods*; D013997:Time Factors
D001549:Benzamides; D004791:Enzyme Inhibitors; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011994:Recombinant Proteins; D009243:NAD; C037689:benzamide; C025160:3-aminobenzamide; D011065:Poly(ADP-ribose) Polymerases