Anti-Poly (ADP-Ribose) Polymerase Protocol

Product No. 11835238001

Protocol

Working concentration

This product is a polyclonal serum and therefore no concentration value for the antibody is provided. The recommended working concentration for western blot analysis is 1.0-0.5 μl/ml in blocking buffer. An antibody dilution up to ten times higher is possible, provided this higher dilution is stored at -15 to -25 °C between applications.

Use in immunoprecipitation

Roche has not tested the application of this product for immunoprecipitations (IP). The typical standard concentration for antisera in IP is 1-5 μl per reaction. A protocol for IP using an anti-PARP antiserum is found in: Dantzer,F., H.P.Nasheuer, J.L.Vonesch, G.de Murcia and J.Menissier-de Murcia (1998). Functional association of poly(ADP-ribose) polymerase with DNA polymerase alpha-primase complex: a link between DNA strand break detection and DNA replication. Nucl Acids Res 26:1891-1898.

Immunofluorescence staining of cryosections

Roche has not tested the application of this antibody on cryosections. There is reason to believe that the antibody will specificially label cryosections, but it is important to note that there will only be specific staining of the nucleus. For this reason, it will not be possible to detect apoptotic cells using this antibody, because one cannot differentiate between the cleaved and uncleaved PARP protein (this is a general statement for any immunofluorescence application using anti-PARP). The anti-PARP antibody recognizes both the 113kD full-length protein as well as the 89 and 24 kD fragments. Detection of PARP cleavage products is therefore only possible using western blot analysis.

Protein quantity for western blot analysis

To resolve the PARP cleavage products, typically 10-50 μg protein per lane should be sufficient to detect the 89 kD and 24 kD PARP cleavage products. When the full-length band (113 kD) is obviously well visible in your western blot experiment but the cleavage products are not, one may suspect that too little apoptosis had been induced (perhaps a longer induction time (e.g., 24 h) would help. Also, we recommend to perform appropriate positive controls, like incubation of U937 cells with 4 μg/ml camptothecin (a potent inducer of apoptosis) for 4 h. Please note that the 24 kD cleavage product is quite often only weakly detected in western blot analysis or even not at all.

Materials

     
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