Facilitate the separation of nuclear extracts and cytoplasmic fractions from the same set of cells in less than two hours

The preparation of good nuclear protein extracts is central to the success of many gene regulation studies. Nuclear extracts are used instead of whole cell lysates for the following reasons. First, many experiments in the area of gene regulation are adversely affected by cellular components present in whole cell lysates. Second, the concentration of the nuclear protein of interest is diluted by the vast array of cytoplasmic proteins present in whole cell extracts. Finally, whole cell lysates are complicated by the presence of genomic DNA and mRNA.

A variety of methods exist to isolate nuclei and prepare nuclear protein extracts.^4-6 Most of these are, however, lengthy processes requiring mechanical homogenization, freeze/thaw cycles, extensive centrifugation or dialysis steps that may compromise the integrity of many fragile nuclear proteins. Pierce developed NE-PER Nuclear and Cytoplasmic Extraction Reagents, which enable a stepwise lysis of cells that generates both functional cytoplasmic and nuclear protein fractions in less than two hours.

Figure 1 shows the results of Micro BCA Protein Assays performed on both the cytosolic and nuclear fractions prepared from a variety of cell lines.

Figure 1. Total protein profile of cytoplasmic and nuclear extracts prepared from a variety of mammalian cell lines using NE-PER Reagents. Protein was quantitated using Pierce Micro BCA Protein Assay Reagent (Product # 23235). Numbers shown are the average of two separate isolations.

• Fast – obtain nuclear and cytoplasmic fractions in less than two hours
• Easy – eliminate freeze/thaw cycles, Dounce homogenization, lengthy centrifugation times and cold room work
• Versatile – obtain nuclear and cytoplasmic extracts separately from the same set of cells or tissue^1,2
• Compatible – with downstream assays, including Western blotting, gel-shift assays, protein assays, reporter gene assays and enzyme activity assays^1-3

Protein Yield

The data show that the yield of protein in the cytoplasmic extract is cell-line dependent, with the larger cell sizes such as HeLa and Hepa 1-6 giving more total protein (500 µg) compared to the smaller NIH3T3 fibroblasts and rat C6 brain cells (250 µg). Nuclear protein yields averaged 100-200 µg total protein from 2 x 10⁶ cells at a concentration on the order of 1.5 mg/ml, independent of cell type. 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.

The key to success for the NE-PER Kit is the stepwise isolation of cytoplasmic and nuclear fractions, which maintains the integrity of the two cellular compartments before extraction. This prevents cross-contamination of proteins between the two fractions.

Figure 2A. Western blots of specific proteins fractionated using NE-PER into cytosolic extracts (CE) and nuclear extracts (NE). 2x106 A549 cells were lysed using Pierce Nuclear and Cytoplasmic Extraction Reagent Kit (NE-PER). Samples were normalized for protein concentration using Pierce BCA Protein Assay. 10 µg of each cytosolic and nuclear extract sample was analyzed by 4-20% SDS-PAGE and Western blotted using specific antibodies diluted 1:1000 (HSP90, GSK3b, RanMEK1, MSH2, MCM2, HDAC1, DDX3, TopoIIb, NFkB) or 1:10000 (p53, GSK3b, b-catenin, GAPDH). Anti-mouse (H+L) HRP (Thermo Scientific) or anti-rabbit (H+L) HRP (Thermo Scientific) diluted 1:25,000 was used as the secondary antibody with Pierce SuperSignal West Dura Chemiluminescent Substrate (Thermo Scientific) for detection.

Equal amounts of A549 cell nuclear and cytoplasmic extracts were Western blotted for known nuclear (DDX3, p53, TOPO IIb, MSH2, MCM2, RAN, HDAC1) and cytoplasmic (HSP90, GAPDH, MEK1, NFkB p65) localized proteins (Figure 2A). Blots show a minimal amount (< 10%) of cross contamination between nuclear and cytoplasmic compartments as determined by densitometric analysis of the films. Two proteins, GSK3b and b-catenin, were found to be equally distributed between nuclear and cytoplasmic compartments.

Figure 2B. Comparison of proteins identified by mass spectrometry in nuclear extract (NE-PER) and whole cell extracts (M-PER) 2 x106 A549 cells were lysed using Mammalian Protein Extraction Reagent (M-PER) or Nuclear and Cytoplasmic Extraction Reagent Kit (NE-PER), and 50 µg of each lysate was separated by 4-20% SDS-PAGE. Gels were stained with Gel Code Blue (Thermo Scientific) before digestion and alkylation of protein peptides using a Pierce In-Gel Tryptic Digest Kit. Peptides were analyzed using a hybrid linear ion trap-Orbitrap mass spectrometer (Finnigan LTQ-FT, Thermo Scientific). Proteins were identified using the BioWorks 3.3.1 software suite (Thermo Scientific) by searching the SEQUEST database (ipi.Human.v3.18.fasta). Identified proteins were manually annotated for function using the Swis-Prot database.

To more broadly assess enrichment of nuclear proteins in NE-PER-derived extracts, proteins isolated from A549 cells using NE-PER or the whole cell lysis buffer, Mammalian Protein Extraction Reagent (M-PER, #78501), were identified by mass spectrometry. The 500 most abundant proteins after each extraction method were manually annotated by cellular function which was used to determine probable cellular localization. As shown in Figure 2B, there was significant increase in nuclear proteins known to be involved in nuclear structure, DNA synthesis, RNA processing/stability and transcription after NE-PER fractionation when compared to M-PER whole cell lysates. There was also a decrease observed in both cytosolic localized microtubule cytoskelatal proteins and cellular metabolism proteins.

A. b-Galactosidase

B. DNA Polymerase

Figure 3. Compartmentalization of cytoplasmic and nuclear fractions is maintained using NE-PER Reagents. A) Extracts from C6 cells (rat glial cells expressing b-galactosidase) were assayed for b-Gal activity using a commercially available kit. B) DNA polymerase activity was assayed using primed M13 single-stranded DNA, buffer, salt and nucleotide composition obtained from the literature. DNA synthesis was monitored by quantitating the amount of [32P]- dCMP incorporated into TCA-precipitable CPMs.

Application: Electrophoretic Mobility Shift Assay

The electrophoretic mobility shift assay (EMSA) is one of the key applications in the area of gene regulation. Nuclear extracts prepared with NE-PER Reagents were used with the LightShift Chemiluminescent EMSA Kit (Product # 20148). The volume of nuclear extract used in these reactions did not exceed 2 µl (10% of total reaction volume). If the protein of interest is less abundant, the concentration of the nuclear extract can be increased by decreasing the volume of NE-PER Extraction Reagent used during extraction. If it still is necessary to use larger volumes of extract for an EMSA, Pierce Slide-A-Lyzer MINI Dialysis Units can be used to remove interfering substances in the NER Nuclear Reagent.

Figure 4. Chemiluminescent EMSA of four different DNA-protein complexes. DNA binding reactions were performed using 20 fmol biotin-labeled DNA duplex (1 biotin per strand) and 2 µl (6.8 µg total protein) NE-PER Nuclear Extract prepared from HeLa cells. For reactions containing specific competitor DNA, a 200- fold molar excess of unlabeled specific duplex was used.

1. Trotter, K. W. and Archer, T. K. (2004) Reconstitution of glucocorticoid receptor-dependent transcription in vivo. Molecular and Cellular Biology 24(8), 3347-3358.
2. Liu, H.B., et al. (2003). Estrogen receptor alpha mediates estrogen’s immune protection in autoimmune disease. J. Immunol. 171(12), 6936-6940.
3. Yamada, N. A. et al. (2003) Variation in the extent of microsatellite instability in human cell lines with defects in different mismatch repair genes. Mutagenesis 18(3), 277-282.
4. Kim, K. et al. (2002) Expression of a functional Drosophila melanogaster N-acetylneuraminic acid (Neu5Ac) phosphate synthase gene: evidence for endogenous sialic acid biosynthetic ability in insects. Glycobiology 12(2), 73-83.
5. Lee, S. and River, C. Role played by hypothalamic nuclear factor-kB in alcohol-mediated activation of the rat hypothalamic-pituitary-adrenal axis. Endocrinology 146(4), 2006-2014.
6. Penheiter, S. G. et al. (2002) Internalization-dependent and -independent requirements for transforming growth factor b receptor signaling via the Smad pathway. Molecular and Cellular Biology 22(13), 4750-4759.
7. Shih, H-P. et al. (2002) Identification of septin-interacting proteins and characterization of the Smt3/SUMO-conjugation system in Drosophila. Journal of Cell Science 115(6), 1259-1271.
8. Yamada, N. A. and Farber, R. A. (2002) Induction of a low level of microsatellite instability by overexpression of DNA polymerase b1. Cancer Research 62, 6061-6064.
9. Adilakshmi, T. and Laine, R.O. (2002). Ribosomal protein S25 mRNA partners with MTF-1 and La to provide a p53-mediated mechanism for survival or death. J. Biol. Chem. 277(6), 4147-4151.
10. Sanceau, J. (2002). Interferons inhibit tumor necrosis factor-a-mediated matrix metalloproteinase-9 activation via interferon regulatory factor-1 binding competition for NF-kb. J. Biol. Chem. 277, 35766-35775.
11. Smirnova, I. V. et al. (2000) Zinc and cadmium can promote rapid nuclear translocation of metal response element-binding transcription factor-1. Journal of Biological Chemistry 275(13), 9377-9384.
12. Current Protocols in Molecular Biology (1993).
13. Zerivitz, K. and Akusjarvi, G. (1989). Gene Anal. Tech. 6, 101-109.
14. Dignam, J.D., et al. (1983). Nucleic Acids Res. 11, 1475-1489.

Ordering Information

Certificate of Analysis   Product Instructions   MSDS

Buy Product # Description Pkg. Size Files Price Add 78833 NE-PER Nuclear and Cytoplasmic Extraction Reagents Sufficient reagents for extracting 50 cell pellet fractions having packed cell volumes of 20 µl each (a total of approx. 2 g of cell paste). Kit includes: Cytoplasmic Extraction Reagent I (CER I), 10 ml Cytoplasmic Extraction Reagent II (CER II), 550 µl Nuclear Extraction Reagent (NER), 5 ml Kit $196.00 Add 78835 NE-PER Nuclear and Cytoplasmic Extraction Reagents Sufficient reagents for extracting 250 cell pellet fractions having packed cell volumes of 20 µl each (a total of approx. 10 g of cell paste). Kit includes: Cytoplasmic Extraction Reagent I (CER I), 50 ml Cytoplasmic Extraction Reagent II (CER II), 2.75 ml Nuclear Extraction Reagent (NER), 25 ml Kit $495.00