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| Product Name | Cleaved PARP (Asp214) ELISA Kit, Apoptosis Marker Quantification |
| Catalog No. | CCAT-HMM-0068 |
| Description | Solid-phase sandwich ELISA kit for quantitative measurement of the 89 kDa cleaved fragment of poly (ADP-ribose) polymerase (PARP) produced by caspase-3 cleavage at Asp214 during apoptosis. PARP, a 116 kDa nuclear enzyme involved in DNA repair, is one of the earliest and most specific substrates cleaved during the execution phase of apoptosis. The ELISA uses a capture antibody specific for the N-terminal 89 kDa cleavage fragment and a detection antibody recognizing a distinct epitope within the same fragment, providing high specificity for the apoptosis-specific PARP cleavage product with no cross-reactivity with full-length PARP (116 kDa) or the 24 kDa C-terminal fragment. |
| Intended Use | Quantitative measurement of apoptosis-specific cleaved PARP (cPARP) levels in cellular lysates as a robust and early biochemical marker of caspase-3-mediated apoptosis for drug efficacy studies, toxicity screening, and mechanism-of-action research. |
| Principle / Technology | Capture antibody specific for N-terminal neoepitope of 89 kDa cPARP fragment (Asp214 cleavage site); detection antibody recognizes distinct epitope within 89 kDa fragment; HRP-conjugated secondary antibody with TMB colorimetric substrate (absorbance 450 nm). |
| Detection Method | Colorimetric microplate reader at 450 nm (reference 570 or 620 nm). |
| Sample Type | Mammalian cell lysates (1×10^5-5×10^6 cells); tissue homogenates. |
| Performance Range / Specifications | Standard curve range: 0.16-10 ng/mL; cell lysate linearity across 0.1-10 µg total protein loaded; intra-assay CV <6%; inter-assay CV <8%. |
| Sensitivity / LOD | Minimum detectable dose: 0.08 ng/mL; detectable in lysates from staurosporine-treated (0.5 µM, 4 hr) Jurkat cells at 0.5 µg protein load. |
| Specificity | Specific for human cleaved PARP 89 kDa fragment; <0.1% cross-reactivity with full-length PARP; does not cross-react with cleaved PARP from mouse or rat (<5% cross-reactivity). |
| Reaction Conditions / Protocol | Load 100 µL standard/sample; incubate 2 hr at RT with shaking; wash 4×; add detection antibody (1 hr); wash; add HRP conjugate (30 min); wash; add TMB (15 min); stop with H2SO4; read at 450 nm. |
| Components / Formulation | Anti-cPARP coated 96-well microplate, cPARP standard (recombinant, 10 ng/vial), detection antibody concentrate, HRP-streptavidin concentrate, assay diluent, wash buffer concentrate (20×), TMB substrate, stop solution (2 N H2SO4), plate sealers, protocol. |
| Storage Conditions | Store at 2-8 °C; do not freeze. |
| Shelf Life | 6 months from date of manufacture. |
| Package Specifications | 1 × 96-well kit (80 samples in duplicate), 5 × 96-well kit. |
| Product Form | Pre-coated microplate; liquid reagents and standards. |
| Quality Control | Each lot tested for standard curve linearity (R² >0.995), spike recovery (85-115%), and detection of cleaved PARP in staurosporine-treated Jurkat cells (signal >5× untreated). |
| Key Features | Specific for apoptosis-cleaved PARP 89 kDa fragment; no cross-reactivity with full-length PARP; quantitative sandwich ELISA format; pre-coated and ready-to-use; TMB colorimetric detection; early apoptosis marker (detectable before DNA fragmentation). |
| Purity | Capture antibody affinity purified; cPARP standard >90% purity by SDS-PAGE. |
| Concentration | cPARP standard: reconstitute to 10 ng/mL then serially dilute; detection antibody: use at 1:200 dilution. |
| Activity / Unit Definition | Capture antibody Kd <0.5 nM for 89 kDa cPARP fragment; HRP conjugate RZ >3.0. |
| Molecular Weight | Cleaved PARP fragment: ~89 kDa (amino acids 1-214). |
| Source / Origin | Rabbit polyclonal anti-cPARP antibody; recombinant human cPARP 89 kDa standard expressed in E. coli. |
| pH Range / Optimal pH | Assay diluent and wash buffer pH 7.2-7.4. |
| Shipping Conditions | Cold pack (2-8 °C). |
| Expiration Date / Stability | 6 months at 2-8 °C; after opening, use within 3 months. |
| Regulatory / Compliance | For research use only; not for diagnostic procedures. |
| Compatibility | Compatible with cell lysates prepared with RIPA, NP-40, or provided lysis buffer. Avoid SDS concentrations >0.1% in sample as it interferes with antibody binding. Protease inhibitor cocktail recommended for sample preparation. |
| Recommended Buffer System | PBS-based assay diluent and wash buffer with Tween-20, pH 7.4. |
| Application Notes / Precautions | Prepare cell lysates on ice with protease inhibitor cocktail. Protein quantification (BCA or Bradford) recommended before ELISA to normalize loading. Avoid freeze-thaw of lysates — aliquot and store at -80 °C. Include staurosporine-treated positive control and untreated negative control lysates. For time-course experiments, collect samples at multiple time points (2, 4, 6, 24 hours) to capture cPARP kinetics. Stop solution addition should produce uniform yellow to blue color change. |
| Batch-to-Batch Consistency | cPARP standard protein content within ±10% of specification; standard curve EC50 within ±20% of reference lot. |
For research use only, not for clinical use.
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