Preparation of Nuclear and Cytoplasmic Extracts from Mammalian Cells

Susan M. Abmayr1, Tingting Yao1, Tari Parmely1, Jerry L. Workman1

1 The Stowers Institute for Medical Research, Kansas City, Missouri
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.3
DOI:  10.1002/0471141755.ph1203s35
Online Posting Date:  December, 2006
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Abstract

Extracts prepared from the isolated nuclei of cultured cells have been instrumental in dissecting the mechanisms by which transcription and mRNA processing occur. These extracts are able to recapitulate accurate transcription initiation and splicing in vitro, which has been useful in direct functional studies. They also serve as the starting material for purification of proteins that can then be reassembled in functional studies or examined in more detail biochemically. This unit describes the preparation of nuclear extracts from cultured cells and optimized production of transcriptionally active extracts from HeLa cells. Additional protocols describe optimization of the method to increase the yield of specific proteins, adaptation of the method for downstream applications such as affinity purification, and preparation of the cytoplasmic (S‐100) fraction.

Keywords: Nuclear extract; cytoplasmic extract

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Nuclear Extracts
  • Support Protocol 1: Optimization of Nuclear Extraction
  • Alternate Protocol 1: Preparation of Extracts for Affinity Purification
  • Basic Protocol 2: Preparation of the Cytoplasmic (S‐100) Fraction
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Nuclear Extracts

  Materials
  • Mammalian (i.e., HeLa) cells from spinner cultures or monolayer cultures
  • Phosphate‐buffered saline (PBS)
  • Hypotonic buffer (see recipe)
  • Low‐salt buffer with 0.02 M KCl (see recipe)
  • High‐salt buffer with 1.2 M KCl (see recipe)
  • Dialysis buffer (see recipe)
  • Liquid nitrogen
  • Beckman JS‐4.2 and JA‐20 rotors (or equivalent)
  • 50‐ml graduated, conical, polypropylene centrifuge tubes (or 15‐ml tubes for smaller extract volumes)
  • Glass Dounce homogenizer with type B (loose) pestle
  • Magnetic stirrer or tiltboard
  • Dialysis membrane tubing (≤14,000 MWCO)
  • Conductivity meter
  • Additional reagents and equipment for Bradford protein assay ( appendix 3A)
NOTE: Perform this procedure at 0° to 4°C, preferably in a cold room. Use precooled buffers and equipment. All centrifugations are performed at 4°C with precooled rotors.

Support Protocol 1: Optimization of Nuclear Extraction

  • High‐salt buffer with 0.8, 1.0, 1.2, 1.4, and 1.6 M KCl
NOTE: As in protocol 1, perform all steps at 0° to 4°C and use precooled buffers and equipment.

Alternate Protocol 1: Preparation of Extracts for Affinity Purification

  • Low‐salt buffer without KCl (see recipe)
  • 5 M NaCl
  • Sterile glycerol (optional)
  • Beckman Type 70.1 Ti rotor (or equivalent)
NOTE: As in protocol 1, perform all steps at 0° to 4°C and use precooled buffers and equipment.

Basic Protocol 2: Preparation of the Cytoplasmic (S‐100) Fraction

  • Cytoplasmic extract (see protocol 1, step )
  • 10× cytoplasmic extract buffer (see recipe)
  • Beckman Type 50 fixed‐angle rotor (or equivalent)
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Figures

Videos

Literature Cited

   Abmayr, S.M., Workman, J.L., and Roeder, R.G. 1988. The pseudorabies immediate early protein stimulates in vitro transcription by facilitating TFIID: Promoter interactions. Genes Dev. 2:542‐553.
   Dignam, J.D., Lebovitz, R.M., and Roeder, R.G. 1983a. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucl. Acids Res. 11:1475‐1489.
   Dignam, J.D., Martin, P.L., Shastry, B.S., and Roeder, R.G. 1983b. Eukaryotic gene transcription with purified components. Methods Enzymol. 101:582‐598.
   Krainer, A.R., Maniatis, T., Ruskin, B., and Green, M.R. 1984. Normal and mutant human beta‐globin pre‐mRNAs are faithfully and efficiently spliced in vitro. Cell 36:993‐1005.
   Lue, N.F. and Kornberg, R.D. 1987. Accurate initiation at RNA polymerase II promoters in extracts from Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 84:8839‐8843.
   Manley, J.L., Fire, A., Cano, A., Sharp, P.A., and Gefter, M.L. 1980. DNA‐dependent transcription of adenovirus genes in a soluble whole‐cell extract. Proc. Natl. Acad. Sci. U.S.A. 77:3855‐3859.
   Sato, S., Tomomori‐Sato, C., Parmely, T.J., Florens, L., Zybailov, B., Swanson, S.K., Banks, C.A., Jin, J., Cai, Y., Washburn, M.P., Conaway, J.W., and Conaway, R.C. 2004. A set of consensus mammalian mediator subunits identified by multidimensional protein identification technology. Mol. Cell. 14:685‐691.
   Schnitzler, G.R. 2000. Isolation of histones and nocleosome cores from mammalian cells. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 22.5.1‐22.5.12. John Wiley & Sons, Hoboken, N.J.
   Weil, P.A., Segall, J., Harris, B., Ng, S.Y., and Roeder, R.G. 1979a. Faithful transcription of eukaryotic genes by RNA polymerase III in systems reconstituted with purified DNA templates. J. Biol. Chem. 254:6163‐6173.
   Weil, P.A., Luse, D.S., Segall, J., and Roeder, R.G. 1979b. Selective and accurate initiation of transcription at the Ad2 major late promotor in a soluble system dependent on purified RNA polymerase II and DNA. Cell 18:469‐484.
Key Reference
   Dignam et al., 1983a. See above.
  This is the original paper describing the preparation of nuclear extracts from HeLa cells. It also optimizes conditions for transcription from multiple class II promoters in these extracts.
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