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
Background
To fully obtain an understanding of cellular processes, fractionation of nuclear and cytoplasmic compartments are needed. There are many protocols and even some commercial kits available to help separate the two compartments. However, most require high centrifugation speeds and there is a high discrepancy in the yield and even the methods to verify the amount of contamination in the final products. Our protocol utilizes a small benchtop centrifuge at low speeds to obtain highly pure extractions for the cytoplasmic and a combined nuclear/perinuclear associated compartments as well as the data analysis to verify the percentage of contamination. To date, the cells lines that have been tested are 293 T, HeLa and GHOST cell lines. (Galvis, 2014; Galvis et al., 2014).
Materials and Reagents
Equipment
Procedure
Data analysis
Notes
Recipes
Acknowledgments
This work was supported in part by grant ID07-I-124 from the California HIV-AIDS Research Program to David Camerini. Support of the UCI Center for Virus Research, UCI Cancer Research Institute and the Chao Family Comprehensive Cancer Center are acknowledged. Alvaro Galvis was supported by the UCI Medical Scientist Training Program, grant T32-GM08620. This protocol is adapted and modified from the original in Sambrook and Russell, 2001.
References
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