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Histone extraction protocol for western blot
- Harvest cells and wash twice with ice-cold PBS. PBS and subsequent buffers can be supplemented with 5 mM sodium butyrate to retain levels of histone acetylation.
- Resuspend cells in Triton Extraction Buffer (TEB: PBS containing 0.5% Triton X 100 (v/v), 2 mM phenylmethylsulfonyl fluoride (PMSF), 0.02% (w/v) NaN3) at a cell density of 107 cells per ml.
- Lyse cells on ice for 10 min with gentle stirring.
- Centrifuge at 6,500 x g for 10 min at 4°C to spin down the nuclei. Remove and discard the supernatant.
- Wash the nuclei in half the volume of TEB and centrifuge as before.
- Re-suspend the pellet in 0.2 N HCl at a density of 4x107 nuclei per ml.
- Acid extract the histones over night at 4°C.
- Centrifuge samples at 6,500 x g for 10 min at 4°C to pellet debris.
- Save the supernatant, which contains the histone protein, and determine protein content using the Bradford assay.
- Store aliquots at -20°C.
View our other epigenetics and western blot related protocols and techniques.