Histone Extraction Protocol

• Dounce homogenizer
• 15 ml conical tubes or 2 ml microcentrifuge tubes
• Centrifuge (capable of supporting 15 ml conical tubes or 2 ml microcentrifuge tubes)
• TEB buffer
• PBS, supplemented with:
• 0.5% Triton X-100
• 2 mM PMSF
• 0.02% NaN3
• Extraction buffer
• 0.5 N HCl
• 10% glycerol
• Acetone
• Distilled water

Protocol

1. Cut the sample into small (1-2 mm3) pieces with a scalpel or scissors.
2. Transfer the pieces to a Dounce homogenizer.
3. Add 1 mL of TEB buffer per 200 mg of tissue.
4. Disaggregate the pieces by 50-60 strokes.
5. Transfer the homogenized mixture to a 15 mL conical tube (or a 2 mL microcentrifuge tube if the total volume is less than 2 mL).
6. Centrifuge at 3,000 rpm for 5 min at 4°C (or at 10,000 rpm for 1 min at 4°C with microcentrifuge tubes).
7. Remove the supernatant.

1. Harvest the cells and pellet by centrifugation at 1,000 rpm for 5 min at 4°C.
2. Resuspend the cells in 1 mL of TEB buffer per 107 cells.
3. Incubate on ice for 10 min with gentle stirring.
4. Centrifuge at 3,000 rpm for 5 min at 4°C.  (If the total volume is less than 2 mL, transfer the cell lysate to a 2 mL microcentrifuge tube and centrifuge at 10,000 rpm for 1 min at 4°C.)
5. Remove the supernatant.

Get News Bulletin Updates

Enjoyed what you read here today? Sign up below and receive updates about new articles, bench tips and protocols so you can stay up-to-date!