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Yeast Histone Preparation II

Method

  1. Grow a 1- to 2-liter culture of diploid yeast to a density of approximately 2 x 108 cells/ml and pellet the yeast by centrifugation in a Beckman JA-10 rotor (or similar) for 5 minutes at 5000 rpm at 4°C.
  2. Resuspend the cell pellet in sterile water (about 200 ml) and centrifuge as above. Discard the supernatant.
  3. Resuspend the cell pellet in 50 ml of 0.1mM Tris, pH 9.4, 10mM DTT. Incubate for 15 minutes at 30°C with gentle shaking.
  4. Centrifuge as above, wash the pelleted cells in 100 ml of Buffer 2 (no protease inhibitors), and re-centrifuge.
  5. Resuspend the cell pellet in 50 ml of Buffer 2 with protease inhibitors. Add protease inhibitors at every step until the histones are extracted in step 10. Add 2 mls of 10 mg/ml zymolyase (dissolved in S Buffer) and incubate at 30°C with gentle shaking.

    Periodically, examine a sample of cells microscopically to determine the optimal length of enzyme treatment. Place a 5- to 10-µl sample of cells on a glass microscope slide and an equal volume of 1% SDS, near to the cells but not touching. Drop a small coverslip onto both droplets and view microscopically (You will need a 40X objective.).


    You should see bright refractile yeast at the edge of the coverslip on the cell droplet side. Where the cell and SDS droplets have run together, you should see a mixture of bright refractile cells and ‘ghosts’. ‘Ghosts’ are yeast cells that have lost their refractile cell wall in the presence of the SDS. They are often slightly larger than the intact yeast but should not be too much larger. They are difficult to see on a regular light microscope and it may take a little experience to see them. They are easy to see on a phase contrast scope.

    Continue the incubation until about 95% of the yeast cells in SDS are ghosted. Typically, this takes 30-60 minutes.

  6. Add of 100 ml of ice-cold Buffer 3, and centrifuge the cells in a JA10 rotor (5 minutes, 4°C, 3.5K).
  7. Resuspend the cell pellets are in 50 ml of ice-cold NIB and hold on ice water for 20 minutes, and spin at 4K in a JA-10 rotor for 5 minutes. Repeat this wash 2X.
  8. Wash the cells 3X in A wash, holding on ice water for 15 minutes for the first two washes.
  9. Wash the cell pellet in Buffer B one time; hold on ice water for 5 minutes. After centrifugation, resuspend the cells again in Buffer B and centrifuge immediately.
  10. Extract the histones by resuspending the pellet in 10 ml of cold 0.4 NH2SO4, and holding in ice water for 30 minutes, vortexing occasionally. Remove debris from the solution by spinning for 10 minutes at 10K

Solutions

S buffer

  • 1.2M Sorbitol
  • 1mM DTT

Buffer 2

  • 1.2M Sorbitol
  • 20mM Hepes pH 7.4
  • 1mM PMSF*
  • 0.5 µg/ml Leupeptin*
  • 0.7 µg/ml Pepstatin*

Buffer 3

  • 1.2M Sorbitol
  • 20mM Pipes
  • 1mM MgCl2, pH 6.8
  • 1mM PMSF*
  • 0.5 µg/ml Leupeptin*
  • 0.7 µg/ml Pepstatin*

NIB

(Nuclei Isolation Buffer)

  • 0.25M Sucrose
  • 60mM KCl
  • 14mM NaCl
  • 5mM MgCl2
  • 1mM CaCl2
  • 15mM MES, pH 6.6
  • 0.8% Triton X-100
  • 0.7 µg/ml Pepstatin*
  • 1mM PMSF*
  • 0.5 µg/ml Leupeptin*

A wash

  • 10mM Tris, pH 8.0
  • 0.5% NP-40
  • 75mM NaCl
  • 30mM Sodium Butyrate*
  • 1mM PMSF*
  • 0.5 µg/ml Leupeptin*

B wash

  • 10mM Tris, pH 8.0
  • 0.4M NaCl
  • 30mM Sodium Butyrate*
  • 1mM PMSF*
  • 0.5 µg/ml Leupeptin*
  • 0.7 µg/ml Pepstatin*


*Add just before use!

Notes:

  1. Haploid yeast may be used. Use of diploid yeast is suggested to increase yield. Protease deficient strains may also increase yield.
  2. Complete resuspension and washing is very important. It is easier to resuspend completely using a small volume, about 10 ml, and a pipet. Add the rest of the volume after the cell pellet is completely resuspended.
  3. Solid sodium butyrate may be used directly. If liquid is used, the pH of the solution must be determined after addition, as butyric acid will change the pH dramatically.

© 2014 The University of Texas MD Anderson Cancer Center