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| Product Name | Cytochrome c ELISA Kit, Mitochondrial Release Detection |
| Catalog No. | CCAT-HMM-0069 |
| Description | Quantitative sandwich ELISA kit for measurement of cytochrome c protein levels in cytoplasmic and mitochondrial cell fractions. During the intrinsic (mitochondrial) apoptosis pathway, cytochrome c is released from the mitochondrial intermembrane space into the cytoplasm where it triggers apoptosome assembly, caspase-9 activation, and downstream executioner caspase cascades. The ELISA enables differential quantification of cytoplasmic vs. mitochondrial cytochrome c by analyzing subcellular fractions prepared from the same cell pellet, providing a quantitative measure of mitochondrial outer membrane permeabilization — a key commitment step in intrinsic apoptosis. |
| Intended Use | Quantitative detection of cytochrome c release from mitochondria to cytoplasm as a marker of intrinsic apoptosis pathway activation in response to DNA damage, growth factor withdrawal, oxidative stress, and chemotherapeutic drug treatment. |
| Principle / Technology | Capture antibody specific for holocytochrome c (heme-attached native form); detection antibody recognizes distinct epitope on cytochrome c; HRP-TMB colorimetric detection at 450 nm; analysis of cytoplasmic and mitochondrial fractions from same sample enables calculation of release percentage. |
| Detection Method | Colorimetric microplate reader: absorbance at 450 nm (reference 570 nm). |
| Sample Type | Cytoplasmic and mitochondrial fractions from cultured mammalian cells (5×10^6 cells minimum); fresh and frozen tissue mitochondrial preparations. |
| Performance Range / Specifications | Standard curve range: 0.31-20 ng/mL; linear across 0.1-15 µg protein loaded; intra-assay CV <7%; inter-assay CV <10%. |
| Sensitivity / LOD | Minimum detectable dose: 0.15 ng/mL; cytochrome c release detectable in as few as 2×10^5 cells after apoptosis induction. |
| Specificity | Recognizes native holocytochrome c (heme-bound) from human, mouse, and rat; <5% cross-reactivity with apocytochrome c (heme-free); no cross-reactivity with cytochrome b, cytochrome b5, or myoglobin. |
| Reaction Conditions / Protocol | Dilute samples in assay diluent; incubate 2 hr at RT; wash 4×; detection antibody 1 hr; wash; HRP-streptavidin 30 min; wash; TMB substrate 15 min; stop; read 450 nm. |
| Components / Formulation | Anti-cytochrome c coated 96-well plate, cytochrome c standard (human recombinant, 20 ng/vial), detection antibody, HRP-streptavidin, assay diluent, wash buffer (20×), TMB substrate, stop solution, cytoplasmic and mitochondrial extraction buffers, protease inhibitor cocktail, plate sealers. |
| Storage Conditions | Store at 2-8 °C; extraction buffers at -20 °C. |
| Shelf Life | 6 months from date of manufacture. |
| Package Specifications | 1 × 96-well kit (includes extraction buffers for 20 samples). |
| Product Form | Pre-coated microplate; liquid reagents; lyophilized standard; extraction buffers. |
| Quality Control | Each lot tested for standard curve linearity (R² >0.99), spike recovery in cell lysates (80-120%), detection of cytochrome c redistribution in staurosporine-treated HeLa cells (cytoplasmic/mitochondrial ratio increase >3-fold vs. untreated). |
| Key Features | Quantitative cytochrome c release measurement; includes subcellular fractionation buffers; detects native holocytochrome c form; cross-species reactivity (human, mouse, rat); pre-coated plate; validated for intrinsic apoptosis pathway research. |
| Purity | Anti-cytochrome c antibody affinity purified; recombinant cytochrome c standard ≥95% purity. |
| Concentration | Cytochrome c standard: reconstitute to 20 ng/mL, then serially dilute. |
| Activity / Unit Definition | Capture antibody affinity: Kd <0.2 nM for holocytochrome c. |
| Molecular Weight | Cytochrome c: ~12.4 kDa (human, 104 amino acids + heme). |
| Source / Origin | Mouse monoclonal capture antibody; recombinant human cytochrome c standard expressed in E. coli with heme reconstitution. |
| pH Range / Optimal pH | Assay buffer pH 7.2-7.4. |
| Shipping Conditions | Cold pack (2-8 °C); extraction buffers may be shipped on dry ice. |
| Expiration Date / Stability | 6 months at recommended storage; extraction buffers stable for 12 months at -20 °C. |
| Regulatory / Compliance | For research use only; not for clinical diagnostic applications. |
| Compatibility | Compatible with cell lysates from human, mouse, and rat origin. Extraction buffers are optimized for cultured cells; for tissue samples, additional homogenization steps may be required. Avoid reducing agents (DTT, β-mercaptoethanol >1 mM) as they may reduce heme iron and affect antibody recognition. |
| Recommended Buffer System | PBS-based assay diluent, pH 7.4; extraction buffers: HEPES-sucrose with protease inhibitors. |
| Application Notes / Precautions | Work quickly and keep all samples and buffers ice-cold during subcellular fractionation. Verify fraction purity by western blot (cytoplasmic marker: β-tubulin; mitochondrial marker: COX IV or VDAC). Normalize cytochrome c concentration to protein content in each fraction. Calculate release percentage: [cyto c]cyto / ([cyto c]cyto + [cyto c]mito) × 100. Include staurosporine (1 µM, 4-6 hr) treated cells as positive control for complete release. |
| Batch-to-Batch Consistency | Standard curve OD values within ±15% of reference lot; spike recovery 80-120% in standardized cell lysates. |
For research use only, not for clinical use.
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