Preparation of Protein Extracts from Yeast

Barbara Dunn1, C. Richard Wobbe2

1 Memorial Sloan‐Kettering Cancer Center, New York, New York, 2 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 13.13
DOI:  10.1002/0471142727.mb1313s23
Online Posting Date:  May, 2001
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Abstract

Three protocols are presented for preparing protein extracts; they differ primarily in the way the cells are broken. In the basic protocol, cells are enzymatically converted to spheroplasts, which are then lysed by a combination of osmotic shock and Dounce homogenization. A support protocol for isolating intact nuclei by differential centrifugation is also presented. An alternate protocol describes mechanical breakage of cells by vortexing in the presence of glass beads. In a second alternate protocol, growing cells are frozen immediately in liquid nitrogen and then lysed by grinding in an industrialā€strength blender in the presence of liquid nitrogen.

     
 
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Table of Contents

  • Basic Protocol 1: Spheroplast Preparation and Lysis
  • Support Protocol 1: Nuclei Preparation by Differential Centrifugation
  • Alternate Protocol 1: Cell Disruption Using Glass Beads
  • Alternate Protocol 2: Cell Disruption Using Liquid Nitrogen
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Spheroplast Preparation and Lysis

  Materials
  • Protease‐deficient yeast cells (BJ926, EJ101, or equivalent)
  • YPD medium (unit 13.1)
  • recipeZymolyase buffer, room temperature and ice‐cold
  • Zymolyase 100T ( ICN Immunobiologicals)
  • 1 M sorbitol (optional)
  • recipeLysis buffer
  • recipeExtraction buffer
  • recipeStorage buffer
  • Liquid nitrogen
  • Sorvall GS‐3 or GSA rotor (or equivalent)
  • Sorvall SS‐34 or SA‐600 rotor (or equivalent)
  • Beckman Type 45Ti rotor (or equivalent; large capacity)
  • 30°C shaker platform
  • Rubber policeman
  • Dounce homogenizer
  • Rotating wheel or rocker
  • Additional reagents and equipment for growing yeast cells (units 13.1 & 13.2), large‐volume 3.NaNdialysis ( 3.NaNappendix 3), and determining conductivity (unit 10.10)

Support Protocol 1: Nuclei Preparation by Differential Centrifugation

  Additional Materials
  • recipeFicoll buffer, ice‐cold
  • Teflon pestle tissue homogenizer, motor‐driven (optional; Thomas)

Alternate Protocol 1: Cell Disruption Using Glass Beads

  Additional Materials
  • recipeGlass bead disruption buffer
  • recipeChilled, acid‐washed glass beads (0.45‐ to 0.55‐mm; see reagents and solutions)
  • Bead Beater and vessel ( Biospec; optional)

Alternate Protocol 2: Cell Disruption Using Liquid Nitrogen

  Additional Materials
  • Yeast cakes (optional; Red Star)
  • Liquid nitrogen
  • 60‐ml syringe
  • 1‐liter plastic beaker (Nalgene or equivalent)
  • 1‐liter stainless steel blender cup and blender (Waring or equivalent)
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Figures

Videos

Literature Cited

Literature Cited
   Amati, B.B. and Gasser, S.M. 1988. Chromosomal ARS and CEN elements bind specifically to the yeast nuclear scaffold. Cell 54:967‐978.
   Bloom, K.S. and Carbon, J. 1982. Yeast centromere DNA is in a unique and highly ordered structure in chromosomes and small circular minichromosomes. Cell 29:305‐317.
   Klekamp, M.S. and Weil, P.A. 1982. Specific transcription of homologous class III genes in yeast Saccharomyces cerevisiae soluble cell‐free extracts. J. Biol. Chem. 257:8432‐8441.
   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.
   Nelson, R.G. and Fangman, W.L. 1979. Nucleosome organization of the yeast 2µ DNA plasmid: A eukaryotic minichromosome. Proc. Natl. Acad. Sci. U.S.A. 76:6515‐6519.
   Sorger, P.K. and Pelham, H.R.B. 1987. Purification and characterization of a heat‐shock element binding protein from yeast. EMBO J. 6:3035‐3041.
   Sorger, P.K., Ammerer, G., and Shore, D. 1989. Identification and purification of sequence‐specific DNA‐binding proteins. In Protein Function: A Practical Approach (T.E. Creighton, ed.) pp. 199‐223. IRL Press. Oxford.
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