Nuclear and Cytoplasmic Protein Extraction Kit, IC-7911
Product Introduction
This product is a ready-to-use kit for the rapid and efficient separation and extraction of cytoplasmic and nuclear protein fractions from cultured cells and tissues. The subcellular compartmentalization of proteins is a fundamental regulatory mechanism in eukaryotic cells, controlling processes such as signal transduction, transcription, apoptosis, and cell cycle progression. The selective extraction of cytoplasmic and nuclear proteins is essential for studying protein translocation events (e.g., NF-κB, p53, STAT proteins), nuclear receptor activation, transcription factor activity, and compartment-specific post-translational modifications. This kit employs a differential detergent-based fractionation method that selectively permeabilizes the plasma membrane to release cytoplasmic proteins while preserving nuclear membrane integrity, followed by nuclear lysis to extract nuclear proteins. The optimized buffer system ensures minimal cross-contamination between fractions and preserves protein integrity and activity. The extracted proteins are compatible with a wide range of downstream applications including Western blot, EMSA, ELISA, and enzymatic activity assays.
Product Features
1. Efficient Fractionation: Differential detergent-based method provides clean separation of cytoplasmic and nuclear protein fractions with minimal cross-contamination.
2. Preservation of Protein Integrity: Optimized buffer system with protease inhibitors maintains protein stability, phosphorylation status, and enzymatic activity.
3. Rapid Protocol: Complete subcellular fractionation achieved in 60-90 minutes from cell pellet or tissue sample.
4. Versatile Sample Compatibility: Validated for use with cultured cells (suspension and adherent) and fresh or frozen tissues.
5. Ready-to-Use Kit Format: Includes all necessary cytoplasmic extraction buffer, nuclear extraction buffer, and protease inhibitor cocktail for a complete workflow.
Specifications
Size: Available in 20 extractions, 50 extractions, and 100 extractions
Sample Types: Cultured cells (suspension and adherent), fresh or frozen tissues
Fractions: Cytoplasmic fraction and nuclear fraction
Downstream Applications: Western blot, EMSA, ELISA, co-immunoprecipitation, enzymatic activity assays
Storage and Stability
Storage Conditions: Store all kit components at 4°C. Protease inhibitor cocktail should be stored at -20°C. Protect from light.
Shelf Life: The product is stable for 12 months from the date of manufacture when stored as directed.
Protocol (For Reference Only)
Important: All steps should be performed at 4°C or on ice. Pre-chill all buffers and tubes. Add protease inhibitors freshly to extraction buffers immediately before use. Minimize the interval between sample harvesting and extraction.
1. Buffer Preparation: Immediately before use, add the provided protease inhibitor cocktail to the required volumes of Cytoplasmic Extraction Buffer A and Nuclear Extraction Buffer B at the recommended ratio. Keep buffers on ice throughout the procedure.
2. Cell Harvesting: For adherent cells, wash cells twice with ice-cold PBS, scrape cells into PBS, and collect by centrifugation at 300 x g for 5 minutes at 4°C. For suspension cells, collect by centrifugation and wash twice with ice-cold PBS. For tissues, mince tissue into small pieces, homogenize in ice-cold PBS, filter through a 70 µm cell strainer, and collect cells by centrifugation.
3. Cytoplasmic Protein Extraction: Resuspend the cell pellet (typically 2-5×10⁶ cells or 20-50 mg tissue) in 200-500 µL of ice-cold Cytoplasmic Extraction Buffer A. Vortex vigorously for 15 seconds to fully resuspend the cell pellet. Incubate on ice for 10-15 minutes with intermittent vortexing. Add the appropriate volume of Cytoplasmic Extraction Buffer B, vortex for 5 seconds, and incubate on ice for 1 minute. Vortex again for 5 seconds, then centrifuge at 12,000-16,000 x g for 5 minutes at 4°C. Immediately transfer the supernatant (cytoplasmic fraction) to a clean pre-chilled tube and store at -80°C.
4. Nuclear Protein Extraction: Wash the remaining pellet (nuclear fraction) once with ice-cold PBS and centrifuge at 12,000 x g for 2 minutes at 4°C. Carefully remove the supernatant without disturbing the pellet. Resuspend the nuclear pellet in 50-100 µL of ice-cold Nuclear Extraction Buffer. Vortex vigorously for 15 seconds, then incubate on ice for 30 minutes with vigorous vortexing for 15 seconds every 10 minutes. Centrifuge at 12,000-16,000 x g for 10 minutes at 4°C. Transfer the supernatant (nuclear fraction) to a clean pre-chilled tube and store at -80°C.
5. Quality Control and Downstream Analysis: Quantify protein concentration in both fractions by BCA or Bradford assay. Verify fraction purity by Western blot using compartment-specific markers: β-tubulin or GAPDH for cytoplasmic fraction, and lamin A/C, histone H3, or nucleolin for nuclear fraction. Use fractions immediately or store at -80°C for long-term storage.
Precautions
1. All steps must be performed at 4°C or on ice to prevent protein degradation and maintain compartment integrity.
2. Add protease inhibitors freshly to extraction buffers immediately before use; buffers without inhibitors should not be stored for extended periods.
3. Optimize buffer volumes based on cell pellet size; insufficient buffer volume results in incomplete extraction, while excessive volume dilutes protein concentration.
4. Verify fraction purity by Western blot using compartment-specific markers; optimize vortexing and incubation times if cross-contamination is observed.
5. For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: How do I verify the purity of the cytoplasmic and nuclear fractions?
A1: Fraction purity is verified by Western blot using compartment-specific antibodies. Recommended markers include β-tubulin or GAPDH for the cytoplasmic fraction, and lamin A/C, histone H3, or nucleolin for the nuclear fraction. Absence of nuclear markers in the cytoplasmic fraction and vice versa indicates clean separation.
Q2: Can I use this kit for tissue samples?
A2: Yes. Fresh or frozen tissues should be minced and homogenized in ice-cold PBS, filtered to remove debris, and processed as described for cell pellets. Tissue samples may require optimized buffer volumes and homogenization conditions for efficient extraction.
Q3: Are the extracted proteins compatible with enzyme activity assays?
A3: Yes. The mild detergent-based extraction preserves protein native conformation and enzymatic activity. However, the inclusion of protease inhibitors and maintenance of low temperatures are essential for preserving activity. Verify compatibility of the extraction buffer components with specific enzymatic assays.
Q4: How can I minimize cross-contamination between fractions?
A4: Ensure thorough washing of the nuclear pellet with PBS after cytoplasmic extraction to remove residual cytoplasmic proteins. Avoid disturbing the pellet when aspirating the cytoplasmic supernatant. Optimize vortexing and incubation times for your specific cell type.
Disclaimer
1. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
2. Due to the variable nature of biological research, optimization of extraction conditions is recommended for specific cell types and tissues.
3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages, including loss of samples or data.
4. Wear appropriate protective clothing and gloves when handling this product.
Ordering Information
Catalog Number: IC-7911
Product Name: Nuclear and Cytoplasmic Protein Extraction Kit
Size: 20 extractions / 50 extractions / 100 extractions
Price:
20 extractions: CNY ¥1250.00 / USD $125.00 / EUR �150.00 / JPY ¥22500.00
50 extractions: CNY ¥1850.00 / USD $185.00 / EUR �222.00 / JPY ¥33300.00
100 extractions: CNY ¥2650.00 / USD $265.00 / EUR �318.00 / JPY ¥47700.00
2023 Version
