Histones are the main protein constituent of chromatin, acting as spools around which DNA winds and compacts. They can undergo various posttranslational modifications (e.g., methylation, acetylation, phosphorylation), which can alter their interaction with DNA and other nuclear proteins and subsequently affect a number of biological processes, including gene expression, DNA repair, and chromosome condensation (mitosis). Thus, extraction methods that can efficiently isolate histones while preserving their modifications are essential in epigenetic studies of these proteins.
Due to the highly basic nature of histone proteins, isolation protocols typically involve acid-based extraction whereby histones are separated from DNA and most other nuclear proteins based on differences in their pH-dependent solubility. Below is an acid extraction protocol for isolating histones from cell/tissue samples.
Materials required
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. For tissues:
Cut the sample into small (1-2 mm3) pieces with a scalpel or scissors.
Transfer the pieces to a Dounce homogenizer.
Add 1 mL of TEB buffer per 200 mg of tissue.
Disaggregate the pieces by 50-60 strokes.
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).
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).
Remove the supernatant.
For cells:
Harvest the cells and pellet by centrifugation at 1,000 rpm for 5 min at 4°C.
Resuspend the cells in 1 mL of TEB buffer per 107 cells.
Incubate on ice for 10 min with gentle stirring.
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.)
Remove the supernatant.
2.Resuspend the pellet in ~200 µL of extraction buffer per 107 cells or per 200 mg of tissue.
3. Incubate on ice for 30 min.
4. Centrifuge at 12,000 rpm for 5 min at 4°C.
5. Transfer the supernatant to a new tube.
6. Add ~0.6 mL of acetone per 107 cells or per 200 mg of tissue.
7. Incubate at -20°C overnight.
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