NP-40 Fractionation and Nucleic Acid Extraction in Mammalian Cells

Alvaro E Galvis; Hugh E Fisher; David Camerini

doi:10.21769/BioProtoc.2584

NP-40 Fractionation and Nucleic Acid Extraction in Mammalian Cells

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

Authors

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

¹ Department of Molecular Biology & Biochemistry, University of California, Irvine, CA, USA.
² Division of Infectious Disease, University of California Irvine, CA, USA.
³ Cancer Research Institute, Irvine CA, USA.
⁴ Center for Virus Research, University of California, Irvine, CA, USA.
⁵ Institute for Immunology, University of California, Irvine, CA, USA.
⁶ Department of Pediatrics, University of Nevada Las Vegas School of Medicine, Las Vegas Nevada, USA.

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.