Nuclear extraction and fractionation

This nuclear extraction and fractionation protocol provides a reliable method for isolating nuclear components from mammalian cells. Optimized for use with cultured cells, the protocol leverages the advantages of cultured cells for reproducibility and experimental control. Designed for researchers preparing samples for western blotting and other downstream applications, the protocol uses a series of centrifugation steps and buffer treatments to separate nuclei from cytoplasmic and membrane fractions. The procedure emphasizes temperature control and mechanical disruption to ensure high-quality nuclear isolation. With clear reagent recipes and step-by-step instructions, this guide supports reproducible results and is compatible with Abcam’s range of antibodies and detection reagents. Whether you’re working with adherent or suspension cells, this protocol offers a streamlined approach to subcellular fractionation for molecular biology research.

Introduction

Nuclear extraction enables the isolation of nuclear proteins for analysis of transcription factors, chromatin-associated proteins, and other nuclear components. Protein extraction is a key step in isolating these nuclear proteins, allowing for efficient and selective recovery for downstream analyses. This protocol provides a simplified process using centrifugation and buffer-based fractionation, ensuring minimal cross-contamination between cellular compartments. The method is optimized for western blotting and other immunodetection assays, making it ideal for researchers studying gene regulation, epigenetics, and signal transduction. By following this protocol, scientists can obtain enriched nuclear fractions suitable for high-sensitivity applications. The guide also includes buffer formulations and handling tips to maintain protein integrity throughout the extraction process.

Background and principles

The principle behind nuclear extraction involves differential centrifugation and selective buffer conditions to separate cellular compartments. Cells are lysed mechanically and chemically, allowing the separation of cytoplasmic and nuclear compartments—cytoplasmic contents are released while nuclear integrity is preserved. Subsequent centrifugation steps isolate the nuclear pellet, which is then washed and resuspended for downstream analysis. The protocol utilizes hypotonic buffers and protease inhibitors to prevent protein degradation and maintain their functionality. In contrast, whole-cell lysis results in a mixture of cellular components, whereas this protocol allows for the selective isolation of the nucleus. Sonication is employed to shear genomic DNA and homogenize the nuclear lysate. This approach ensures that nuclear proteins, as well as other molecules such as DNA and RNA from the nuclear fraction, are extracted in a form suitable for western blotting, immunoprecipitation, and other analytical techniques. Isolating the nucleus is crucial for downstream molecular biology applications that require pure nuclear fractions.

Stage 1 - Procedure

All centrifugation should be done at 4°C. Samples should be kept on ice throughout the procedure.

Steps

Transfer cells from 10 cm plates into 500 μL fractionation buffer, eg by scraping. Incubate for 15 min on ice.

• Just before use, add the following per 10 mL:

Using a 1 mL syringe, pass the cell suspension through a 27-gauge needle 10 times (or until all cells are lysed).

Leave on ice for 20 min.

Centrifuge the sample at 720 x g for 5 min.

• The pellet will contain the nuclei, and the supernatant will contain cytoplasm, membrane, and mitochondria.

Wash the nuclear pellet remaining after Step 4 with 500 μL of fractionation buffer.

• Disperse the pellet with a pipette and pass it through a 25-gauge needle 10 times.
• Centrifuge again at 720 x g for 10 min.
• Discard the supernatant and keep the pellet containing the nuclei.

Resuspend the pellet in TBS with 0.1% SDS.

• Sonicate the suspension briefly to shear genomic DNA and homogenize the lysate (3 sec on ice at a power setting of 2).

• Comparison to other methods

  Compared to whole-cell lysis protocols like RIPA buffer extraction, this nuclear fractionation method offers superior compartmental specificity. While RIPA disrupts all cellular membranes, resulting in mixed fractions, this protocol preserves nuclear integrity during initial lysis, enabling cleaner separation. Commercial kits may simplify the process but often lack transparency in reagent composition, making troubleshooting difficult. This protocol provides detailed buffer recipes and mechanical steps, allowing researchers to understand and control each stage. Additionally, this method avoids harsh detergents that can denature proteins, making it more suitable for functional studies of nuclear proteins and transcription factors.

• Applications

  This nuclear extraction protocol is widely applicable in molecular biology and biomedical research. It is particularly useful for preparing samples for western blotting, chromatin immunoprecipitation (ChIP), and transcription factor analysis. Researchers studying gene expression, epigeneticregulation, or nuclear signaling pathways can benefit from the enriched nuclear fractions obtained. This protocol can be adapted for use with various cell types, including adherent and suspension cultures. Whether investigating disease mechanisms or validating biomarkers, this protocol provides a robust foundation for analyzing nuclear proteins.

• Limitations

  While effective, this protocol has limitations that users should consider. Mechanical lysis using needles and sonication requires precision to avoid incomplete disruption or protein degradation. The method is optimized for mammalian cells and may require adaptation for other organisms. Additionally, centrifugation steps must be carefully timed and temperature-controlled to prevent sample loss or contamination. The use of SDS in the final resuspension step may not be suitable for all downstream applications, particularly those requiring native protein conformations. Researchers should validate the protocol for their specific experimental needs and consider alternative buffers if necessary.

• Troubleshooting

  Common issues in nuclear extraction include low yield, contamination with cytoplasmic proteins, and protein degradation. To improve yield, ensure thorough mechanical lysis using the recommended needle gauge and sonication settings. Keep samples consistently on ice to prevent proteolysis. If cytoplasmic contamination occurs, verify centrifugation speeds and durations, and repeat washing steps. Use freshly prepared buffers with protease inhibitors to maintain protein integrity. Incomplete lysis may result from insufficient needle passes or inadequate buffer volume. Adjust these parameters according to the cell type and density. For downstream issues like a poor western blot signal, confirm antibody specificity and sample concentration.