78833-- NE-PER Nuclear and Cytoplasmic Extraction Reagents

NE-PER Nuclear and Cytoplasmic Extraction Reagents  

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. The nuclear protein extraction method 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. The isolated proteins can be used to perform immunoassays as well as protein interaction studies such as mobility shift assays （EMSA）, co-immunoprecipitation （Co-IP） and pull-down assays.

• Fast – obtain nuclear and cytoplasmic fractions in less than two hours
• Proven – the NE-PER Reagent Kit is referenced in more than 950 distinct publications
• 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

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 （Part No. 23235）. Values are the average of two separate isolations.

Western blots of specific proteins fractionated using NE-PER into cytosolic extracts （C） and nuclear extracts （N）. Two million 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. 10µg of each cytosolic and nuclear extract sample was analyzed by 4-20% SDS-PAGE and Western blotted using specific antibodies diluted 1:1000 （HSP90, Ran, MEK1, MSH2, MCM2, HDAC1, DDX3, TopoIIb, NFκB） or 1:10000 （p53, GSK3β, β-catenin, GAPDH）. Anti-mouse （H+L） HRP or anti-rabbit （H+L） HRP diluted 1:25,000 was used as the secondary antibody with SuperSignal West Dura Chemiluminescent Substrate for detection.

Total protein profile of cytoplasmic and nuclear extracts prepared from different mouse tissues. Swiss Webster mouse tissues （40mg） were harvested, rinsed with PBS and lysed using the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit. Extracts were quantified using the Pierce 660nm Protein Assay Reagent （Part No. 22660）. Values are the average of two separate isolations.

Western blots of specific proteins from fractionated tissues. Cytoplasmic and nuclear extract （10µg each） from different mouse tissue fractionated using the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit was analyzed by 4-20% SDS-PAGE and Western blotting. Primary antibodies specific for the target proteins were diluted 1:1,000 （SP1, HDAC2 and NFκB p65, or 1:10,000 （GAPDH）. Anti-Rabbit （H+L） HRP （Part No. 31460） diluted 1:25,000 was the secondary antibody and SuperSignal West Dura Chemiluminescent Substrate （Part No. 34076） was used for signal detection.

Chemiluminescent EMSA of four different DNA-protein complexes. DNA binding reactions were performed using 20fmol biotin-labeled DNA duplex （1 biotin per strand） and 2µL （6.8µg 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.

• Trotter, K. W. and Archer, T. K. （2004）. Reconstitution of glucocorticoid receptor-dependent transcription in vivo. Molecular and Cellular Biology 24（8）, 3347-3358.
• Liu, H.B., et al. （2003）. Estrogen receptor alpha mediates estrogen’s immune protection in autoimmune disease. J. Immunol. 171（12）, 6936-6940.
• Yamada, N. A. et al. （2003）. Variation in the extent of microsalite instability in human cell lines with defects in different mismatch repair genes. Mutagenesis 18（3）, 277-282.
• 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.
• Lee, S. and River, C. Role played by hypothalamic nuclear factor-κB in alcohol-mediated activation of the rat hypothalamic-pituitary-adrenal axis. Endocrinology 146（4）, 2006-2014.
• Penheiter, S. G. et al. （2002）. Internalization-dependent and -independent requirements for transforming growth factor β receptor signaling via the Smad pathway. Molecular and Cellular Biology 22（13）, 4750-4759.
• 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.
• Yamada, N. A. and Farber, R. A. （2002）. Induction of a low level of microsalite instability by overexpression of DNA polymerase β1. Cancer Research 62, 6061-6064.
• 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.
• Sanceau, J. （2002）. Interferons inhibit tumor necrosis factor-a-mediated matrix metalloproteinase-9 activation via interferon regulatory factor-1 binding competition for NF-κB. J. Biol. Chem. 277, 35766-35775.
• 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.
• Current Protocols in Molecular Biology （1993）.
• Zerivitz, K. and Akusjarvi, G. （1989）. Gene Anal. Tech. 6, 101-109.
• Dignam, J.D., et al. （1983）. Nucleic Acids Res. 11, 1475-1489.

Related Resources: NE-PER Product FAQ Remove detergent from protein samples Protein stability and storage Related Products: LightShift Chemiluminescent EMSA Kit Micro BCA Protein Assay Kit SuperSignal West Pico Chemiluminescent Substrate Slide-A-Lyzer MINI Dialysis Unit 2-D Sample Preparation for Nuclear Proteins

Ordering Information

Product # Description Pkg. Size Instructions MSDS CofA Price 78833 NE-PER Nuclear and Cytoplasmic Extraction Reagents Formulation: Multi-component kit Sufficient For: 50 samples each containing 2 million cells （20µL packed） Kit Contents: Cytoplasmic Extraction Reagent I （CER I）, 10mL Cytoplasmic Extraction Reagent II （CER II）, 0.55mL Nuclear Extraction Reagent （NER）, 5mL   15mL kit Local contact 78835 Formulation: Multi-component kit Sufficient For: 250 samples each containing 2 million cells （20µL packed） Kit Contents: Cytoplasmic Extraction Reagent I （CER I）, 50mL Cytoplasmic Extraction Reagent II （CER II）, 2.75mL Nuclear Extraction Reagent （NER）, 25mL   75mL kit Local contact