The kit contains a primary monoclonal antibody that detects only the cleaved portion of human PARP, a goat anti-mouse secondary antibody conjugated to DyLight™ 549 Fluorophore and various other reagents and buffers that are required for immunofluorescence staining for high-content screening (HCS) assays.
Poly (ADP-ribose) polymerase (PARP) cleavage is an important marker of caspase 3-mediated apoptosis. PARP is a 116 kDa nuclear protein involved in repair of DNA nicks induced by various stressors and is one of the substrates for caspase 3, which cleaves PARP into a 85 kDa fragment during apoptosis. In human PARP, cleavage occurs at Asp 214 and Gly 215 leading to formation of 89 and 24 kDa fragments. Cleavage of PARP correlates with DNA fragmentation and other morphological changes making it a critical marker of apoptosis.
Cleaved PARP in HeLa cells treated with staurosporine was quantitatively assayed using the Cellomics Cleaved PARP Detection Kit, Cellomics ArrayScan HCS Reader and the Cellomics Compartmental Analysis BioApplication Software Module. Induction of apoptosis by staurosporine leads to cleavage of PARP and, therefore, increased staining in the nucleus by the anti-cleaved PARP antibody. Cells labeled using this kit also can be imaged by fluorescence or confocal microscopy.
Figure 2. Staining of cleaved PARP in HeLa cells treated with vehicle (0.1% DMSO in media)(left) or with 1 µM staurosporine for 3 hours (right). Cells were stained according to the kit protocol and imaged using a Cellomics ArrayScan HCS Reader. Dose response curve for cleaved PARP intensity in treated HeLa Cells (bottom). Wells were scored using the output parameter of the average nuclear intensity of cleaved PARP (Mean_CircAvgInt). Data represents mean ± SD from three plates (eight wells per 96-well plate per dose of staurosporine). EC50 = 0.21 ± 0.08 µM.
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