All tags Western blot Histone extraction protocol for western blot

Histone extraction protocol for western blot

A guide to the preparation of histone proteins for electrophoresis.

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  1. 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. 
  2. 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.
  3. Lyse cells on ice for 10 min with gentle stirring.
  4. Centrifuge at 6,500 x g for 10 min at 4°C to spin down the nuclei. Remove and discard the supernatant.
  5. Wash the nuclei in half the volume of TEB and centrifuge as before.
  6. Re-suspend the pellet in 0.2 N HCl at a density of 4x107 nuclei per ml.
  7. Acid extract the histones over night at 4°C.
  8. Centrifuge samples at 6,500 x g for 10 min at 4°C to pellet debris.
  9. Save the supernatant, which contains the histone protein, and determine protein content using the Bradford assay.
  10. Store aliquots at -20°C.



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