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| Product Name | Mitochondrial Isolation Kit, Density Gradient Method, Cell and Tissue |
| Catalog No. | ATR-SPS-0027 |
| Description | Density gradient centrifugation kit for rapid and high-purity isolation of intact functional mitochondria from cultured cells and fresh tissue samples. The kit employs a discontinuous Percoll gradient method that separates mitochondria from other organelles (lysosomes, peroxisomes, endoplasmic reticulum microsomes) based on buoyant density differences. The optimized homogenization buffer gently disrupts plasma membranes while maintaining mitochondrial outer membrane integrity. Isolated mitochondria retain coupled respiration and membrane potential for downstream functional assays including oxygen consumption, ATP synthesis, calcium uptake, and cytochrome c release measurements. |
| Intended Use | Isolation of intact, functional mitochondria from cultured cells, liver, heart, brain, and skeletal muscle tissue for mitochondrial respiration, membrane potential, calcium handling, protein import, and apoptosis studies. |
| Principle / Technology | Gentle Dounce homogenization to break plasma membrane while preserving mitochondria; differential centrifugation removes nuclei and debris; discontinuous Percoll gradient separates mitochondria (density ~1.13 g/mL) from lysosomes (~1.10) and microsomes (~1.09-1.11). |
| Detection Method | Homogenize tissue in ice-cold isolation buffer using Dounce homogenizer; centrifuge 600 × g, 10 min (remove nuclei); centrifuge supernatant 7,000 × g, 10 min (pellet crude mitochondria); resuspend in 15% Percoll; layer on discontinuous Percoll gradient (23%/40%); centrifuge 30,700 × g, 10 min; collect mitochondria at 23%/40% interface; wash twice. |
| Sample Type | Fresh cultured cells (minimum 5×10^7 cells); fresh liver, heart, brain, kidney, or skeletal muscle tissue (50-500 mg). |
| Performance Range / Specifications | Mitochondrial yield: 0.5-5 mg mitochondrial protein per gram tissue depending on source; purity: >95% mitochondria by VDAC or COX IV western blot; <5% contamination by lysosomal (LAMP1), peroxisomal (catalase), or ER (calnexin) markers; respiratory control ratio (RCR) >4 with glutamate/malate. |
| Sensitivity / LOD | Isolation of mitochondria from as few as 1×10^7 cultured cells; detectable cytochrome c oxidase activity in as little as 1 µg mitochondrial protein. |
| Specificity | Percoll gradient specifically enriches for intact mitochondria based on buoyant density; damaged mitochondria with compromised outer membrane partition to higher density fractions and are excluded. |
| Reaction Conditions / Protocol | All steps performed at 0-4 °C to maintain mitochondrial integrity; isolation complete within 2-3 hours from tissue homogenization to purified mitochondrial suspension. |
| Components / Formulation | Homogenization buffer (5× concentrate), Percoll solution (sterile), 10× Mitochondrial Wash Buffer, Dounce homogenizer compatible protocol, BSA (fatty acid-free, 1 g), protease inhibitor cocktail. |
| Storage Conditions | Store buffers at 2-8 °C; Percoll at 2-8 °C (do not freeze); BSA at 2-8 °C. |
| Shelf Life | 12 months from date of manufacture. |
| Package Specifications | 1 kit (sufficient for ~20 mitochondrial isolations from cultured cells or ~10 from tissue samples). |
| Product Form | Liquid buffers and Percoll suspension; Dounce homogenizer not included. |
| Quality Control | Each lot tested for mitochondrial yield from rat liver tissue, purity by western blot (VDAC enrichment, calnexin depletion), and functional integrity by Clark-type oxygen electrode (RCR >4 with glutamate + malate). |
| Key Features | Density gradient method for high-purity mitochondria; preserves respiratory function; suitable for cells and tissues; Percoll-based non-ionic gradient; complete kit with optimized buffers; ~2-3 hour protocol. |
| Purity | Percoll: sterile colloidal silica particles (15-30 nm) coated with PVP; endotoxin tested; BSA fatty acid-free, ≥98%. |
| Concentration | 5× Homogenization Buffer: 1.5 M sucrose, 50 mM HEPES, 5 mM EGTA, pH 7.4; dilute to 1× before use. |
| Activity / Unit Definition | Isolated mitochondria maintain State 3 respiration (ADP-stimulated) for ≥2 hours on ice; RCR >4 indicates coupled mitochondria. |
| Molecular Weight | Not applicable — multi-component isolation kit. |
| Source / Origin | Percoll from silica sol; synthetic buffer components; BSA from bovine plasma. |
| pH Range / Optimal pH | Homogenization and wash buffers pH 7.4; Percoll gradient pH 7.0-7.4. |
| Shipping Conditions | Cold pack (2-8 °C). |
| Expiration Date / Stability | 12 months at 2-8 °C; Percoll stable at 2-8 °C — do not freeze. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic applications. |
| Compatibility | Compatible with liver, heart, brain, kidney, and skeletal muscle from mouse, rat, and human biopsy tissue. Optimized for fresh tissue — frozen tissue yields poorly coupled mitochondria. Cultured cells: HeLa, HEK293, HepG2, primary hepatocytes, C2C12 myotubes. |
| Recommended Buffer System | Sucrose-HEPES-EGTA homogenization buffer; sucrose-MOPS wash buffer. |
| Application Notes / Precautions | Work quickly and maintain all buffers, tubes, and equipment at 0-4 °C. Use fresh tissue within 30 minutes of harvest. Avoid over-homogenization (10-15 gentle Dounce strokes) as this ruptures mitochondria. Check mitochondrial purity by measuring citrate synthase activity or COX IV western. For functional assays, use within 2 hours of isolation. NADH (1 mM) can help maintain mitochondrial membrane potential during storage. |
| Batch-to-Batch Consistency | Buffer osmolarity within ±10 mOsm; Percoll density within ±0.01 g/mL; mitochondrial yield and purity verified per lot. |
For research use only, not for clinical use.
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