NE-PER™ Nuclear and Cytoplasmic Extraction Reagents, 75 mL - FAQs

Nuclear and cytoplasmic proteins can be isolated within 2 hours.

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After extracting the nuclear proteins using the NER Reagent, genomic DNA will be located in the pellet.

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EDTA-free Halt Protease and/or Phosphatase Inhibitor Cocktails (e.g., Cat. No. 78425) can be added to CER I and NER reagents. Most commercially available protease inhibitor cocktails are also compatible; however, avoid protease inhibitors that contain alcohols. Because the concentration of the CER I and NER Reagents are critical, do not dilute these reagents by more than 5% when adding protease inhibitors.

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No. Also, nuclear protein was not successfully isolated from yeast cells that were pre-lysed with Y-PER Yeast Protein Extraction Reagent.

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Yes. The nuclear fraction can be dialyzed in a Slide-A-Lyzer MINI Dialysis Unit (Cat. No. 69550) against a buffer, such as PBS, that is compatible with the specific downstream application. A buffer exchange can also be performed using the Thermo Scientific Protein Desalting Spin Columns (Cat. No. 89849) or Zeba Desalt Spin Columns (Cat. No. 89882).

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Freezing and thawing will prematurely rupture cellular membranes and lead to high cross-contamination between the nuclear and cytoplasmic fractions.

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Yes. The kit was optimized for a packed-cell volume; therefore, adherent cells require harvesting, either by scraping or trypsinization, from the culture flask for maximum recovery.

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We have extracted soluble nuclear proteins from calf liver tissue and cultured epithelial, fibroid, kidney, liver and brain cells. Many references have cited the use of various other cells and tissues with the NE-PER Reagents.

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The total amount of protein recovered will vary with different sample types. Some examples are listed in the table below:

Cell/Tissue Type, Cytoplasmic Protein, Nuclear Protein
100 mg of liver tissue, 3 to 4 mg, 1 to 1.5 mg
10^6 HeLa cells, 300 to 400 µg, 40 to 60 µg
10^6 monocytes, N/A, 0.15 to 0.25 µg

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Typically, cross-contamination between the nuclear and cytoplasmic fraction is 10% or less when using this kit. Careful pipetting and performing the incubation and centrifugation as indicated in the instructions will minimize cross-contamination. Performing an additional wash of the nuclear pellet with phosphate buffer before adding the NER Reagent will reduce carryover of the cytoplasmic proteins to the nuclear protein fraction.

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Cross-contamination of the nuclear fraction by cytoplasmic proteins can be determined by probing for a protein known to reside only in the cytoplasm, such as a heat shock protein 90 (Hsp90), b-galactosidase or protein kinase C. Determine contamination of the cytoplasmic fraction by probing for Oct-1 or p53 nuclear proteins in the cytoplasmic fraction.

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The following applications were successfully used with protein extracted using the NE-PER Reagents: electrophoretic mobility shift assay (EMSA), Western blotting, reporter assays, isoelectric focusing (IEF) and immunoprecipitation.

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The CER I Reagent induces swelling of the cell causing stress on the cellular membrane. The CER II reagent gently lyses the cell membrane allowing cytoplasmic proteins to be collected, while leaving the nucleus intact. The NER Reagent is then used to extract nuclear proteins from the pellet.

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We do not recommend using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Cat. No. 78833, 78835) with FFPE samples. The extraction process with this product relies on the ability to separate nuclei from the rest of the cell/tissue components and then salt-extract proteins through the nuclear pores to get a ''nuclear protein fraction''. With fixed FFPE samples, this product would not be able to isolate nuclei and/or extract proteins. In addition, reversion of formalin crosslinks in FFPE samples does not guarantee that the integrity of the nuclear membrane and therefore, the compartmentalization of the proteins will be maintained.

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We have not performed any in-house testing for use of NE-PER Nuclear and Cytoplasmic Extraction Reagents (Cat. No. 78833, 78835) for the extraction and fractionation of nucleic acids. However, it is possible that it may work for this application. Please note that this product is not guaranteed to be RNase or DNase free so, you would need to use RNase inhibitors.

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Our R&D team has used p53, Ran, MCM2, MSH2, and/or TOPO II beta as markers for the nuclear fraction. On the other hand, GADPH, HSP90 or MEK1 can be used as markers for the cytoplasmic fraction. Bear in mind that protein expression might slightly vary between tissues and/or biological conditions. In addition, some common nuclear markers (DDX3, HDAC1 or H3) or cytoplasmic markers (GSK3 beta, HfkB P65) can also be found to some extent in the opposite fraction.

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This has not been tested.

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Internal tests with Cox iV, a mitochondrial membrane protein, showed that almost all lysosome and mitochondrial proteins, extracted with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Cat. 78835), were found in the cytosolic fraction, and minimal amount were found in the nuclear fraction when compared to other nuclear/cytoplasmic reagents. There are high density and low-density mitochondria, so we typically see some mitochondria marker proteins in both fractions, but the majority is in the cytoplasmic fraction.

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We offer the following kits for cell fractionation:

Mem-PER Plus Membrane Protein Extraction Kit, Cat. No. 89842
NE-PER Nuclear and Cytoplasmic Extraction Reagents, Cat. No. 78835
Subcellular Protein Fractionation Kit for Cultured Cells, Cat. No. 78840
Subcellular Protein Fractionation Kit for Tissues, Cat. No. 87790
Mitochondrial Isolation Kit for Cultured Cells, Cat. No. 89874
Mitochondrial Isolation kit for Tissue, Cat. No. 89801
Lysosome Enrichment Kit for Tissues and Cultured Cells, Cat. No. 89839

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Isolation of subcellular fractions and concentration of low-abundantce proteins allows for more efficient identification and study of the proteins of interest.

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