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| Product Name | Nuclear and Cytoplasmic Protein Extraction Kit, NE-PER Based, Rapid Protocol |
| Catalog No. | ATR-SPS-0028 |
| Description | Rapid nuclear and cytoplasmic protein extraction kit based on the NE-PER (nuclear and cytoplasmic extraction reagents) principle for sequential detergent-based fractionation of cultured cells and tissues. The kit uses a two-step protocol: first, a mild nonionic detergent (CER I/CER II) selectively permeabilizes the plasma membrane to release cytoplasmic proteins while keeping the nuclear envelope intact. Nuclei are then pelleted and lysed with a high-salt nuclear extraction buffer (NER) to release soluble nuclear proteins. The entire protocol is completed in under 2 hours on ice, avoiding the lengthy centrifugation times of traditional Dounce homogenization methods. |
| Intended Use | Sequential extraction of cytoplasmic and nuclear protein fractions from cultured cells and tissues for western blotting, EMSA, transcription factor activity assays, protein-protein interaction studies, and subcellular localization analysis. |
| Principle / Technology | CER I/II mild detergent system selectively permeabilizes plasma membrane (cholesterol-rich) but not nuclear envelope (cholesterol-poor); cytoplasmic proteins released into supernatant; pelleted nuclei lysed in high-salt NER to extract DNA-binding proteins; compatible with downstream assays. |
| Detection Method | Harvest and wash cells with PBS; add CER I buffer, vortex, incubate 10 min on ice; add CER II, vortex, incubate 1 min on ice; centrifuge 16,000 × g, 5 min, 4 °C; collect supernatant (cytoplasmic fraction); wash nuclear pellet with CER I; resuspend in NER buffer with vigorous vortexing (15 sec every 10 min for 40 min); centrifuge 16,000 × g, 10 min; collect supernatant (nuclear fraction). |
| Sample Type | Fresh or frozen cultured cells (1×10^6-1×10^7 cells); fresh or frozen tissue samples (20-100 mg). |
| Performance Range / Specifications | Protein yield: cytoplasmic fraction 60-80% of total; nuclear fraction 20-40% of total; typical yield 50-200 µg cytoplasmic protein and 10-50 µg nuclear protein per 1×10^6 HeLa cells. |
| Sensitivity / LOD | Detectable by western blotting using 5-20 µg protein load per lane; fraction purity >90% by marker analysis (cytoplasmic: β-tubulin, GAPDH; nuclear: histone H3, lamin A/C, PARP). |
| Specificity | Plasma membrane selective permeabilization preserves nuclear envelope integrity; cross-contamination typically <10% as verified by organelle-specific marker western blots. |
| Reaction Conditions / Protocol | CER I: 10 min, CER II: 1 min; NER: 40 min intermittent vortexing; all steps at 4 °C; total protocol time ~90 minutes. |
| Components / Formulation | CER I buffer (10 mL), CER II buffer (0.55 mL), NER buffer (5 mL), protease inhibitor cocktail (100×), phosphatase inhibitor cocktail (100×), detailed protocol. |
| Storage Conditions | Store buffers at 2-8 °C; inhibitors at -20 °C. |
| Shelf Life | 12 months from date of manufacture. |
| Package Specifications | 1 kit (sufficient for ~50 extractions from 2×10^6 cells each), 5 kits. |
| Product Form | Liquid buffers; DMSO-based inhibitor cocktails (100×). |
| Quality Control | Each lot tested for fractionation efficiency and purity using HeLa cell standard (cytoplasmic marker: GAPDH; nuclear marker: HDAC1 or Lamin B1; cross-contamination <10%). |
| Key Features | Rapid 2-step protocol (~90 minutes); no Dounce homogenizer or ultracentrifuge required; serum-free, detergent-based permeabilization; high-purity cytoplasmic and nuclear fractions; inhibitors included; scalable for 10^5-10^7 cells. |
| Purity | Buffer components molecular biology grade; inhibitor cocktails validated against broad panel of proteases and phosphatases. |
| Concentration | CER I: ready-to-use; CER II: ready-to-use; NER: ready-to-use. |
| Activity / Unit Definition | Extraction efficiency: >80% of GAPDH in cytoplasmic fraction; >70% of histone H3 in nuclear fraction. |
| Molecular Weight | Not applicable — multi-component detergent and salt buffer system. |
| Source / Origin | Synthetic detergents and analytical grade buffer salts. |
| pH Range / Optimal pH | All buffers pH 7.4-7.9. |
| Shipping Conditions | Cold pack or ambient; inhibitors on dry ice recommended. |
| Expiration Date / Stability | 12 months at recommended storage; after adding inhibitors, buffers stable for 1 week at 2-8 °C. |
| Regulatory / Compliance | For research use only; not for diagnostic procedures. |
| Compatibility | Compatible with western blotting (add SDS loading buffer directly), EMSA, ELISA, transcription factor DNA-binding assays, and co-immunoprecipitation (dilute NER 2-5 fold to reduce salt). Optimized for mammalian cells and tissues. Also compatible with Drosophila, C. elegans, and yeast with protocol modifications. |
| Recommended Buffer System | CER I/II: HEPES-based with MgCl2, KCl, EDTA, and nonionic detergents; NER: HEPES-based with high NaCl, MgCl2, EDTA, and glycerol. |
| Application Notes / Precautions | Always add fresh protease and phosphatase inhibitors to buffers before use. Pre-chill all buffers and keep samples on ice throughout. For tissue: mince tissue finely and homogenize in CER I using a Dounce homogenizer (not provided). Avoid over-vortexing during CER steps as this may rupture nuclei. The nuclear pellet should be white and compact after CER extraction — a loose or translucent pellet indicates insufficient detergent permeabilization. For phospho-protein analysis, add phosphatase inhibitors and NaF to NER. |
| Batch-to-Batch Consistency | Buffer composition within specification; fractionation purity >90% for each marker in standardized HeLa cell assay per lot. |
For research use only, not for clinical use.
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