Isolation of Nuclei and Nuclear Membranes From Animal Tissues

John M. Graham1

1 Liverpool John Moores University, Liverpool
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.10
DOI:  10.1002/0471143030.cb0310s12
Online Posting Date:  November, 2001
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Abstract

The vast majority of methods for the isolation of nuclei and nuclear membranes use soft mammalian tissues (particularly rat liver) as the biological source; however, the protocols described in this unit should be applicable generally to any tissue type, cultured cells, or cells of lower eukaryotes or plants, so long as a suitable homogenization method is available. Generally, a buffered isoosmotic medium containing KCl and MgCl2 is used to stabilize the nuclei of mammalian tissues. Some of the media used for cultured animal cells and nonmammalian sources are also described. After homogenization the nuclei are purified using a sucrose barrier of 2.3 M. The use of OptiPrep overcomes the viscosity and osmolarity problems associated with the use of high molarity sucrose, and it permits the use of a high‐speed centrifuge and much shorter centrifugation times. OptiPrep is also used isolate plant cell (i.e., wheat germ) nuclei, because it is able to resolve nuclei from the denser starch granules.

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of Nuclei from Rat‐Liver Homogenate Using a Sucrose Density Barrier
  • Alternate Protocol 1: Isolation of Nuclei from Animal or Plant (Wheat Germ) Cells Using an Iodixanol Gradient
  • Alternate Protocol 2: Isolation of Nuclei from Rat‐Liver Homogenate in a Nonaqueous Medium
  • Basic Protocol 2: Isolation of Nuclear Membranes: High‐Ionic‐Strength Method
  • Basic Protocol 3: Isolation of Nuclear Membranes: Low‐Ionic‐Strength Method
  • Support Protocol 1: Dipheylamine Assay for DNA
  • Support Protocol 2: Orcinol Assay for RNA
  • Support Protocol 3: Ethidium Bromide Assay for DNA and RNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Nuclei from Rat‐Liver Homogenate Using a Sucrose Density Barrier

  Materials
  • 150‐ to 200‐g Sprague‐Dawley rats
  • Nuclear isolation medium (NIM; see recipe)
  • Sucrose density barrier (SDB; see recipe)
  • Scissors or razor blade
  • ∼30‐ml Potter‐Elvehjem homogenizer with 0.1‐mm clearance and loose‐fitting pestle
  • 75‐µm pore size nylon gauze or cheesecloth (muslin)
  • Glass rod
  • Refrigerated low‐speed centrifuge with swinging‐bucket rotor and appropriate 50‐ml polypropylene or polystyrene tubes
  • ∼30‐ml Dounce homogenizer with loose‐fitting Wheaton Type B pestle
  • Refractometer
  • Ultracentrifuge with swinging‐bucket rotor (e.g., Beckman SW40Ti, Sorvall TH641) and appropriate 14‐ml tubes
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.NOTE: All solutions, glassware, centrifuge tubes, and equipment should be precooled to 0° to 4°C, and kept on ice throughout. Centrifuge rotors should be precooled to the same temperature.NOTE: Protease inhibitors (unit 3.4) may be added to any or all solutions.

Alternate Protocol 1: Isolation of Nuclei from Animal or Plant (Wheat Germ) Cells Using an Iodixanol Gradient

  • Filtered homogenate (animal cells or tissue; see protocol 1) or wheat germ
  • 50% (w/v) iodixanol (see recipe; animal cells or tissue)
  • 30% and 35% (w/v) iodixanol gradient solutions (see recipe; animal cells or tissue)
  • 25% and 40% (w/v) iodixanol gradient solutions (see recipe; wheat germ)
  • Wheat germ medium (WGM; see recipe)
  • High‐speed centrifuge with a swinging‐bucket rotor and appropriate 50‐ml tubes
NOTE: All solutions, glassware, centrifuge tubes, and equipment should be precooled to 0° to 4°C, and kept on ice throughout. Centrifuge rotors should be precooled to the same temperature.NOTE: Protease inhibitors (unit 3.4) may be added to any or all solutions.

Alternate Protocol 2: Isolation of Nuclei from Rat‐Liver Homogenate in a Nonaqueous Medium

  • Ethylene glycol homogenization medium (EGHM; see recipe)
  • 10% and 40% (w/v) Nycodenz (Axis‐Shield; available from Accurate Chemicals) in recipeEGHM
  • Two‐chamber gradient maker or Gradient Master (BioComp Instruments)
NOTE: All solutions, glassware, centrifuge tubes, and equipment should be precooled to 0° to 4°C, and kept on ice throughout. Centrifuge rotors should be precooled to the same temperature.NOTE: Protease inhibitors (unit 3.4) may be used in any or all solutions.

Basic Protocol 2: Isolation of Nuclear Membranes: High‐Ionic‐Strength Method

  Materials
  • 200 mM PMSF ( appendix 2A)
  • Nuclei suspension medium (NSM; see recipe)
  • DNase I
  • RNase
  • Incubation buffer (IB; see recipe)
  • High‐NaCl buffer (HNB; see recipe)
  • 2‐mercaptoethanol
  • Nuclear membrane storage medium (NMSM; see recipe)
  • ∼20‐ml Dounce homogenizer with loose‐fitting Wheaton Type B pestle
  • Refrigerated low‐speed centrifuge with swinging‐bucket rotor

Basic Protocol 3: Isolation of Nuclear Membranes: Low‐Ionic‐Strength Method

  Materials
  • 200 mM PMSF ( appendix 2A)
  • Low‐ionic‐strength suspension medium (LISM; see recipe)
  • 1.0 mg/ml DNase I
  • Sucrose buffer, pH 7.4 and 8.5 (SB7.4 and SB8.5; see recipe)
  • NMSM (see recipe)
  • ∼10‐ml Dounce homogenizer with loose‐fitting Wheaton Type B pestle
  • High‐speed centrifuge with fixed‐angle rotor for 15‐ml tubes
NOTE: All centrifugations are carried out at 4°C and all incubations are at 22°C.

Support Protocol 1: Dipheylamine Assay for DNA

  Materials
  • 500 µg/ml DNA standard solution
  • 5%, 10%, and 20% (w/v) trichloracetic acid (TCA), ice‐cold
  • Samples
  • Diphenylamine reagent (see recipe)

Support Protocol 2: Orcinol Assay for RNA

  Materials
  • 500 µg/ml DNA and RNA standard solutions
  • 5%, 10%, and 20% (w/v) TCA
  • Samples
  • Orcinol reagent (see recipe)

Support Protocol 3: Ethidium Bromide Assay for DNA and RNA

  Materials
  • Phosphate‐buffered saline with Ca2+ and Mg2+ (PBSCM; see recipe)
  • 25 µg/ml DNA standard solution: store at –20°C for 2 to 3 months
  • 25 µg/ml heparin
  • Sample
  • 50 µg/ml RNase: heat at 100°C for 10 min to denature DNase
  • 25 µg/ml ethidium bromide
  • 3‐ml cuvettes
  • Spectrophotofluorometer with an emission wavelength of 580 nm and an excitation wavelength of 360 nm
CAUTION: Ethidium bromide is a mutagen and should be handled, stored, and disposed of with appropriate care.
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Figures

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Literature Cited

Literature Cited
   Birnie, G.D. 1978. Isolation of nuclei from animal cells in culture. Methods Cell Biol. 17:13‐26.
   Blobel, G. and Potter, V.R. 1966. Nuclei from rat liver: An isolation method that combines purity with high yield. Science 154:1662‐1665.
   Brunk, C.F., Jones, K.C., and James, T.W. 1979. Assay for nanogram quantities of DNA in cellular homogenates. Anal. Biochem. 92:497‐500.
   Dunphy, W. and Rothman, J.E. 1985. Compartmental organization of the Golgi stack. Cell 40:463‐472.
   Ford, T.C., Baldwin, J.P., and Lambert, S.J. 1998. Rapid enzyme‐free preparation of starch‐free nuclei from plants facilitates studies of chromatin. Plant Protein Club Annual Symposium, p. 24. York, U.K.
   Goldberg, D.E. and Kornfeld, S. 1983. Evidence for extensive subcellular organization of asparagine‐linked oligosaccharide processing and lysosomal enzyme phosphorylation. J. Biol. Chem. 258:3159‐3165.
   Gollin, S.M. and Wray, W. 1984. Isopycnic centrifugation of metaphase chromosomes in Nycodenz. Exp. Cell. Res. 152:204‐211.
   Graham, J.M. and Ford, T.C. 1983. Enzymic and chemical assays compatible with iodinated density gradient media. In Iodinated Density Gradient Media: A Practical Approach (D. Rickwood, ed.), pp. 195‐216. Oxford University Press, Oxford.
   Graham, J., Ford, T., and Rickwood, D. 1994. The preparation of subcellular organelles from mouse liver in self‐generated gradients of iodixanol. Anal. Biochem. 220:367‐373.
   Gurney, T. and Foster, D.N. 1977. Nonaqueous isolation of nuclei from cultured cells. Methods Cell Biol. 16:45‐68.
   Harris, J.R. and Agutter, P.S. 1976. The isolation and characterization of the nuclear envelope. In Biochemical Analysis of Membranes (A.H. Maddy, ed.), pp. 132‐173. Chapman and Hall, London.
   Harris, J.R. and Milne, J.F. 1974. A rapid procedure for the isolation and purification of rat liver nuclear envelope. Biochem. Soc. Trans. 2:1251‐1253.
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   Kay, R.R., Fraser, D., and Johnston, I.R. 1972. A method for the rapid isolation of nuclear membranes from rat liver. Eur. J. Biochem. 30:145‐154.
   Marsh, M., Schmid, S., Kern, H., Harms, E., Male, P., Mellman, I., and Helenius, A. 1987. Rapid analytical and preparative isolation of functional endosomes by free flow electrophoresis. J. Cell Biol. 104:875‐886.
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   Provost, J.J., Fudge, J., Israelit, S., Siddiqi, A.R., and Exton, J.H. 1996. Tissue‐specific distribution and subcellular distribution of phospholipase D in rat: Evidence for distinct RhoA‐ and ADP‐ ribosylation factor (ARF)‐regulated isoenzymes. Biochem. J. 319:285‐291.
   Rickwood, D., Messent, A., and Patel, D. 1997. Isolation and characterization of nuclei and nuclear subfractions. In Subcellular Fractionation: A Practical Approach (J.M. Graham and D. Rickwood, eds.), pp 71‐105. IRL Press at Oxford University Press, Oxford.
   Schneider, W.C. 1957. Determination of nucleic acids in tissues by pentose analysis. Methods Enzymol. 3:680‐684.
   Widnell, C.C. and Tata, J.R. 1964. A procedure for the isolation of enzymically active rat liver nuclei. Biochem. J. 92:313‐317.
   Wray, W., Conn, P.M., and Wray, V.P. 1977. Isolation of nuclei using hexylene glycol. Methods Cell Biol. 16:69‐86.
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