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A Further Step in Understanding Apoptosis,,, Direct Detection of PARP Cleavage

easily employed in many different applications such as flow cytometry, immunocytochemistry, and immunohistochemistry (also in conjunction with an anti-fluorescein antibody). Particularly, in regard to tissue sections, the antibody is a valuable tool in acquiring additional information on the results achieved using the TUNEL method (even in dual-labeling applications).

Typical Experiment
Flow cytometric analysis of PARP cleavage and TUNEL reactivity

HeLa cells were induced to undergo apoptosis via stimulation with anti-CD95 (clone 2R2, 0.2 g/ml) and cycloheximide (10 g/ml). After 5 hours, the untreated cells (Figure 1, left panel) and CD95-stimulated cells (Figure 1, right panel) were stained for PARP cleavage using the anti-PARP Caspase Cleavage Product-Fluorescein antibody (1 g/ml), and analyzed for TUNEL staining using the In Situ Cell Death Detection Kit, TMR red.
Cells in the two right boxes of the dot-blot analysis display PARP-1 cleavage; the upper two boxes indicate TUNEL-positive cells (Figure 1, right panel). Note that PARP cleavage precedes DNA degradation and TUNEL labeling. The staining pattern of the apoptotic small PARP fragment in HeLa cells is also shown by immunofluorescence in Figure 2a. A peptide competition control experiment is included (Figure 2b).

To gain new insights into apoptotic pathways particularly in situ Roche Applied Science now offers the anti- PARP Caspase Cleavage Product-Fluorescein antibody. The antibody can serve as an early marker of apoptosis since it is directed against the caspase cleavage site of PARP-1 and specifically recognizes an epitope that is generated by caspases, but is not accessible in nonapoptotic cells.
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