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. 2017 Oct 20;7(20):e2584.
doi: 10.21769/BioProtoc.2584.

NP-40 Fractionation and Nucleic Acid Extraction in Mammalian Cells

Alvaro E Galvis  1   2   3   4   5   6 , Hugh E Fisher  1   2   3   4   5 , David Camerini  1   2   3   4   5
Affiliations

NP-40 Fractionation and Nucleic Acid Extraction in Mammalian Cells

Alvaro E Galvis et al. Bio Protoc. .

Abstract

This technique allows for efficient, highly purified cytoplasmic and nuclear-associated compartment fractionation utilizing NP-40 detergent in mammalian cells. The nuclear membrane is not disturbed during the fractionation thus leaving all nuclear and perinuclear associated components in the nuclear fraction. This protocol has been modified from Sambrook and Russell (2001) in order to downscale the amount of cells needed. To determine the efficiency of fractionation, we recommend using qPCR to compare the subcellular compartments that have been purified with equivalent amount of control whole cell extracts.

Keywords: Cell fractionation; Cytoplasmic and nuclear extractions; Nucleic acid purification; Quantitative PCR.

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Figures

Figure 1.
Figure 1.. Sample data analysis of fractionation protocol.
This is a ratio of mitochondrial and β-globin DNA in nuclear and cytoplasmic fractions vs. total cell lysates. GHOST-R5X4 cells were transfected either with DBR1 shRNA pHyper-D4 (D4) or DBR1 shRNA triple mismatch, pHyper-M4 (M4). Two to twenty-four hours later, the cells were either fractionated into nuclear and cytoplasmic fractions or lysed to prepare whole cell extracts. DNA was isolated for qPCR to evaluate the success of fractionation. A. β-Globin primers were utilized to assay the exclusion of nuclear contamination in the cytoplasmic fraction in contrast to whole cell extracts. The graph illustrates the ratio of β-globin copies in the cytoplasmic fractions to β-globin copies in the whole cell lysates. B. β-Globin primers were utilized to assay concentration of nuclear fractions compared to whole cell extracts. The graph illustrates the ratio of β-globin copies in the nuclear fractions to β-globin copies in the whole cell lysate. C. Mitochondrial primers were used to assay concentration of cytoplasmic fraction compared to whole cell extracts. The graph illustrates the ratio of mitochondrial DNA copies in the nuclear fractions to β-globin copies in the whole cell lysates. D. Mitochondrial primers were utilized to assay the exclusion of mitochondrial contamination in the nuclear extractions.
See this image and copyright information in PMC

References

    1. Galvis A. E.(2014). An RNA lariat intermediate in HIV-1 cDNA synthesis. ProQuest 3615193.
    1. Galvis A. E., Fisher H. E., Nitta T., Fan H. and Camerini D.(2014). Impairment of HIV-1 cDNA synthesis by DBR1 knockdown. J Virol 88(12): 7054-7069. - PMC - PubMed
    1. Sambrook J. and Russell D. W.(2001). Molecular cloning: a laboratory manual. CSHL Press.