Histone extraction protocol for western blot |
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Save time with an optimized extraction kit To get your histones extracted perfectly the first time, we recommend our Histone Extraction Kit ab113476. Alternatively, we recommend the following protocol. 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 650 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 650 x g for 10 min at 4°C to pellet debris.Save the supernatant (which contains the histone protein) and neutralise HCl with 2M NaOH at 1/10 of the volume of the supernatant.Determine protein content using the Bradford assay.Store aliquots at -20°C.Review Histone extraction kit ab113476 or view our other epigenetics and western blot related protocols and techniques. |
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