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Yeast Histone Preparation I

Method

Day 1

Inoculate 5 ml culture in YPD (or selective media) and grow overnight with shaking at 30°C.

Day 2

Inoculate 1-2 L of YPD (or selective media) with 0.5 ml overnight culture. Grow with shaking at 30°C.

Day 3

Dilute aliquot of cells 1/10 and count:

  • For preparative experiments, cell densities can be high: ~ 2x108/ml
  • For analytical experiments, cell density should reflect log growth (~2x107/ml)

In Steps 5-12, add fresh protease inhibitors leupeptin and pepstatin to all buffers.

  1. Spin cells at 3000 g for 5 min at 4°C.
  2. Wash cell pellets in 200-400 ml sterile water and centrifuge again.
  3. Resuspend cells in 50 ml DTT/Tris-HCl, pH 9.5. Incubate with shaking at 30°C, 15 minutes. Spin as above.
  4. Resuspend pellet in 50 ml Sorbitol/Hepes buffer (Buffer #2). Re-spin.
  5. Resuspend pellet in 50 ml Buffer #2 and add 1-2 ml zymolyase (10 mg/ml). Incubate at 30°C for 45-60 minutes or until well ghosted.
  6. Add 100 ml ice-cold Buffer #3 (Sorbitol/Pipes/MgCl2). Spin at 3.5K (JA10 rotor) for 5 minutes at 4°C.
  7. Resuspend pellet in 50 ml ice-cold NIB buffer and hold on ice for 20 min. Spin at 4K (JA10 rotor) for 5 minutes at 4°C.
  8. Repeat NIB wash 2X, and hold each for 20 minutes on ice.
  9. Resuspend pellet in 50 ml A wash and hold on ice for 15 minutes. Spin as above for 5 minutes.
  10. Repeat A wash and spin.
  11. Resuspend pellet in 50 ml B wash and hold on ice for 5 minutes. Spin as above for 5 minutes.
  12. Resuspend in 25 ml B wash and spin immediately.
  13. Resuspend pellet in 10 ml cold 0.8M H2SO4 to extract histones. Hold on ice for 30 minutes, vortexing occasionally.
  14. Spin at 10K for 10 min. Save supernatant, which contains the extracted histones. Measure volume with pipette.
  15. Add 100% TCA to a final concentration of 20%. A large precipitate should form almost immediately. Hold on ice for 30 minutes.
  16. Spin at 12K for 30 minutes. If supernatant still looks cloudy, pour into a fresh tube and spin again. Wash pellet in 10 ml acid acetone (acetone + 0.1% concentrated HCl) and spin for 5 min at 10K. Wash pellet in cold acetone and spin again at 10K 5 minutes. Pour off acetone and air-dry pellet.
  17. Resuspend and combine pellets in a total of 1 ml of 10 mM Tris-HCl, pH 8.0. If pellet is hard to resuspend, add more Tris buffer, 0.5 ml at time. Try to keep histones as concentrated as possible.
  18. Store histones at 20°C. Run 15 and 30 µl on a 22% SDS-PAGE gel.

Buffers

Store these buffers at RT except NIB and Buffer #3, which should be stored at 4°C for ease of use.

DTT / Tris

  • 0.1 mM Tris-HCl, pH 9.4
  • 10 mM DTT

Buffer 2

  • 1.2 M Sorbitol
  • 20 mM Hepes, pH 7.4

Buffer 3

  • 1.2 M Sorbitol
  • 20 mM Pipes, pH 6.8
  • 1 mM MgCl2

NIB

  • 0.25 M Sucrose
  • 60 mM KCl
  • 15 mM NaCl
  • 5 mM MgCl2
  • 1 mM CaCl2
  • 15 mM MES, pH 6.6
  • 1 mM PMSF
  • 0.8% Triton X-100

A Wash

  • 10 mM Tris-HCl, pH 8.0
  • 0.5 % NP-40
  • 75 mM NaCl
  • 30 mM Sodium Butyrate
  • 1 mM PMSF

B Wash

  • 10 mM Tris-HCl, pH 8.0
  • 0.4 M NaCl
  • 30 mM Sodium Butyrate
  • 1 mM PMSF

© 2014 The University of Texas MD Anderson Cancer Center