NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific™)

The Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Kit provides for efficient cell lysis and extraction of separate cytoplasmic and nuclear protein fractions in less than two hours.

Features of the NE-PER Nuclear and Cytoplasmic Extraction Kit:

• Fast—obtain nuclear and cytoplasmic fractions of soluble proteins in less than two hours
• Proven—the NE-PER Reagent Kit is referenced in more than 950 distinct publications
• Versatile – nuclear protein from either cultured cells or tissues (intended for fresh samples only)
• Scalable—two kit sizes for producing extracts from cells and tissues
• Convenient—simple instructions do not require ultracentrifugation over gradients
• Compatible—use for downstream assays, including Western blotting, gel-shift assays, protein assays, reporter gene assays and enzyme activity assays

The NE-PER Kit is a nuclear protein extraction method that involves simple, stepwise lysis of cells and centrifugal isolation of nuclear and cytoplasmic protein fractions. A benchtop microcentrifuge, tubes and pipettors are the only tools required. The NE-PER Reagents efficiently solubilize and separate cytoplasmic and nuclear proteins into fractions with minimal cross-contamination or interference from genomic DNA and mRNA. Once desalted or diluted, the isolated proteins can be used to perform immunoassays and protein interaction experiments, such as mobility shift assays (EMSA), co-immunoprecipitation (Co-IP) and pull-down assays.

There are a variety of methods available to isolate nuclei and prepare nuclear protein extracts. Most of the methods for preparing nuclear extracts are lengthy processes requiring mechanical homogenization, freeze/thaw cycles, extensive centrifugation or dialysis steps that may compromise the integrity of nuclear proteins. The NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit is a reagent-based protocol that enables the stepwise lysis of cells, separation of the cytoplasm from the intact nuclei and then extraction of nuclear proteins away from genomic DNA and mRNA. This gentle process takes less than two hours and requires only a standard bench top centrifuge when using cultured cells. Furthermore, both active nuclear proteins and cytoplasmic proteins can be recovered from the same cell culture or tissue sample.

From two million cells, the kit typically yields 200 to 500 μg cytoplasmic protein and 100 to 200 μg nuclear protein (at a concentration of 1 mg/mL). Typical cross-contamination between cytosolic and nuclear fractions is about 10%. The method recovers primarily soluble proteins from the nucleus, not so much proteins bound in chromatin or integral membrane proteins of the nuclear envelope. The protein concentration of the nuclear extracts can be manipulated easily by varying the volume of nuclear extraction reagent (NER) used in the extraction without any significant loss in extraction efficiency. Specific extraction of nuclear proteins from cells is central to the success of many gene regulation studies. While a variety of methods exist to isolate nuclei and prepare nuclear protein extracts, most of these are lengthy processes requiring mechanical homogenization, freeze/thaw cycles, extensive centrifugation or dialysis steps that may compromise the integrity of many fragile nuclear proteins. The NE-PER Kit overcomes these problems, providing high-quality, concentrated nuclear protein extracts for use in EMSA and other analysis techniques.

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