ab139410 is an In-Cell ELISA (ICE) assay kit that uses quantitative immunocytochemistry to measure levels of the larger (89 kDa) fragment of PARP protein in cultured cells. Cells are fixed in a microplate and target of interest is detected with highly specific, well-characterized antibody. Relative target levels are quantified using a HRP labeled secondary antibody and a standard spectrophotometer capable reading at 450 nm. Optionally, the antibody signal can be normalized to the total cell stain Janus Green.
In-Cell ELISA (ICE) technology is used to perform quantitative immunocytochemistry on cultured cells with a horseradish peroxidase detector antibody. The technique generates quantitative data with specificity similar to Western blotting, but with much greater quantitative precision and higher throughput due to the greater dynamic range and linearity of the method and the ability to run up to 96 samples in parallel. This method rapidly fixes the cells in situ, stabilizing the in vivo levels of proteins and their post-translational modifications, and thus essentially eliminates changes during sample handling, such as preparation of protein extracts. Finally, the signal can be normalized to cell amount, measured by the provided Janus Green whole cell stain, to further increase the assay precision.
Plates are available in our ICE (In-Cell ELISA) Support Pack (ab111542) which can be bought seperately.
PARP is a 113 kDa nuclear DNA-repair enzyme that transfers ADP-ribose units from NAD+ to variety of nuclear proteins including topoisomerases, histones and PARP itself. Via poly ADP ribosylation, PARP is responsible for regulation of cellular homeostasis including cellular repair, transcription and replication of DNA, cytoskeletal organization and protein degradation. In response to DNA damage, PARP activity is increased upon binding to DNA strand nicks and breaks. Excessive DNA damage leads to generation of large branched ADP-ribose polymers and activation of a unique cell death program. During apoptosis, PARP is cleaved by activated caspase-3 between Asp214 and Gly215, resulting in the formation of an N-terminal 24 kDa fragment containing most of the DNA binding domain and a C-terminal 89 kDa fragment containing the catalytic domain. The proteolysis of PARP through this cleavage renders the enzyme inactive and this further facilitates apoptotic cell death. Thus the presence of 89 kDa PARP fragment is considered to be a very reliable biomarker of apoptosis.
Involved in the base excision repair (BER) pathway, by catalyzing the poly(ADP-ribosyl)ation of a limited number of acceptor proteins involved in chromatin architecture and in DNA metabolism. This modification follows DNA damages and appears as an obligatory step in a detection/signaling pathway leading to the reparation of DNA strand breaks. Mediates the poly(ADP-ribosyl)ation of APLF and CHFR. Positively regulates the transcription of MTUS1 and negatively regulates the transcription of MTUS2/TIP150. With EEF1A1 and TXK, forms a complex that acts as a T-helper 1 (Th1) cell-specific transcription factor and binds the promoter of IFN-gamma to directly regulate its transcription, and is thus involved importantly in Th1 cytokine production. Required for PARP9 and DTX3L recruitment to DNA damage sites. PARP1-dependent PARP9-DTX3L-mediated ubiquitination promotes the rapid and specific recruitment of 53BP1/TP53BP1, UIMC1/RAP80, and BRCA1 to DNA damage sites.
Phosphorylated by PRKDC and TXK. Poly-ADP-ribosylated by PARP2. Poly-ADP-ribosylation mediates the recruitment of CHD1L to DNA damage sites. S-nitrosylated, leading to inhibit transcription regulation activity.
Nucleus. Nucleus, nucleolus. Localizes at sites of DNA damage.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
Dynamic range of the ab139410 assay kit
Dynamic range of the ab139410 assay kit. HeLa cells were allowed attach overnight and treated for 4 hours with 1 µM staurosporine (ab120056). The data, presented as background-subtracted OD 450 nm values (mean +/-SEM), were obtained using this kit as described in the protocol.
Example of determination of staurosporine EC50 of the PARP cleavage using ab139410 assay kit
Example of determination of staurosporine EC50 of the PARP cleavage using ab139410 assay kit. HeLa cells, seeded at 50,000 per well were allowed attach overnight and treated for 6 hours with staurosporine (ab120056) as indicated. The data, presented as Janus Green normalized background-subtracted OD 450 nm values (mean +/-SEM), were obtained using this kit as described. The determined staurosporine EC50 value is 0.28 µM.
Cleaved PARP antibody specificity demonstrated by immunocytochemistry
Cleaved PARP antibody specificity demonstrated by immunocytochemistry. HeLa cells were treated with vehicle (A, B and C) or 1 µM Staurosporine (ab120056, D, E, and F) for 4 hours to induce apoptosis. The cells were treated, permeabilized, blocked and incubated with the Cleaved PARP antibody as described in the protocol of this kit. Samples were further processed for fluorescence immunocytochemistry and co-stained with DNA stain DAPI. Images of green Cleaved PARP signals (A and D), red DAPI signals (B and E) and overlay of these green and red images (C and F) are shown. Note that the Cleaved PARP antibody specifically labels nuclei of staurosporine-treated cells.
Cleaved PARP antibody specificity demonstrated by Western blotting
Figure 4. Cleaved PARP antibody specificity demonstrated by Western blotting. Western blot analysis of HeLa cells treated with vehicle (DMSO, lane 1 and 3) or 1 µM staurosporine (lanes 2 and 4) for 4 hours to induce apoptosis. Blots were incubated with an antibody that recognizes both the full-length PARP-1 and its 89 kDa fragment (left panel), or with the Cleaved PARP antibody used in this kit (right panel). Appropriate HRP-conjugated secondary antibodies followed by ECL detection were used. Note that the Cleaved PARP antibody recognizes the apoptosis-specific 89 kDa fragment of PARP but it does not recognize the 113 kDa full-length PARP.
Specificity of the ab139410 assay kit
Specificity of the ab139410 assay kit. HeLa cells, seeded at indicated densities, were allowed attach overnight and treated for 4 hours with drug vehicle (DMSO) or 1 µM staurosporine (ab120056) to induce apoptosis. The data (mean +/-SEM), presented as background-subtracted OD 450 nm values (A), or Janus green-normalized signals (B), were obtained using this kit as described in the protocol.