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.
- Spin cells at 3000 g for 5 min at 4°C.
- Wash cell pellets in 200-400 ml sterile water and centrifuge again.
- Resuspend cells in 50 ml DTT/Tris-HCl, pH 9.5. Incubate with shaking at 30°C, 15 minutes. Spin as above.
- Resuspend pellet in 50 ml Sorbitol/Hepes buffer (Buffer #2). Re-spin.
- 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.
- Add 100 ml ice-cold Buffer #3 (Sorbitol/Pipes/MgCl2). Spin at 3.5K (JA10 rotor) for 5 minutes at 4°C.
- 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.
- Repeat NIB wash 2X, and hold each for 20 minutes on ice.
- Resuspend pellet in 50 ml A wash and hold on ice for 15 minutes. Spin as above for 5 minutes.
- Repeat A wash and spin.
- Resuspend pellet in 50 ml B wash and hold on ice for 5 minutes. Spin as above for 5 minutes.
- Resuspend in 25 ml B wash and spin immediately.
- Resuspend pellet in 10 ml cold 0.8M H2SO4 to extract histones. Hold on ice for 30 minutes, vortexing occasionally.
- Spin at 10K for 10 min. Save supernatant, which contains the extracted histones. Measure volume with pipette.
- Add 100% TCA to a final concentration of 20%. A large precipitate should form almost immediately. Hold on ice for 30 minutes.
- 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.
- 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.
- 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