A Histone Extraction Protocol

histone extraction protocol

The following histone extraction protocol was written by scientists at Epigentek and is a recommended and optimized procedure for their histone modification assays, successfully utilized by many research labs. Use these easy to follow steps to ensure proper isolation of histone proteins.

  1. For tissues (treated and untreated), weigh the sample and cut the sample into small pieces (1-2 mm3) with a scalpel or scissors. Transfer tissue pieces to a Dounce homogenizer.  Add TEB buffer (PBS containing 0.5% Triton X 100, 2 mM PMSF and 0.02% NaN3) at 1 ml per 200 mg of tissue, and disaggregate tissue pieces by 50-60 strokes. Transfer homogenized mixture to a 15 ml conical tube and centrifuge at 3000 rpm for 5 minutes at 4°C. If total mixture volume is less than 2 ml, transfer mixture to a 2 ml vial and centrifuge at 10,000 rpm for 1 minute at 4°C. Remove supernatant.

For cells (treated and untreated), harvest cells and pellet the cells by centrifugation at 1000 rpm for 5 minutes at 4°C. Resuspend cells in TEB buffer at 107 cells/ml and lyse cells on ice for 10 minutes with gentle stirring. Centrifuge at 3000 rpm for 5 minutes at 4°C. If total volume is less than 2 ml, transfer cell lysates to a 2 ml vial and centrifuge at 10,000 rpm for 1 minute at 4°C. Remove supernatant.

  1. Resuspend cell/tissue pellet in 3 volumes (approx. 200 µl/107 cells or 200 mg tissues) of extraction buffer (0.5N HCl + 10% glycerol) and incubate on ice for 30 minutes.
  1. Centrifuge at 12,000 rpm for 5 minutes at 4°C and remove the supernatant fraction to a new vial.
  1. Add 8 volumes (approx. 0.6 ml/107 cells or 200 mg tissues) of acetone and leave at –20°C overnight.
  1. Centrifuge at 12,000 rpm for 5 minutes and air-dry the pellet. Dissolve the pellet in distilled water (30-50 µl/107 cells or 200 mg tissues).
  1. Quantify the protein concentration. Aliquot the extract and store the extract at –20°C or –80°C.

If you’re looking for a commercial kit, Epigentek also offers the EpiQuik Total Histone Extraction Kit for extracting histone proteins in just one hour.

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  • Mohammed Ahmed

    What do u mean at the first step when you mention( Add TEB buffer

    (PBS containing 0.5% Triton X 100, 2 mM PMSF and 0.02% NaN3)

    at 200 mg/mL) what do u meen with at 200mg/ml

    • mastercytosine

      We’ve updated this protocol for clarity! Should have been 1 ml per 200 mg of tissue!

  • Juozas B

    What do you mean in second step 0.5N ? What is N?

    • mastercytosine

      Hi Juozas, N stands for normality, which in this case is the same as 0.5M HCl — good luck!

  • Hany

    Hi,
    thank you for the protocol!
    I followed the protocol however some samples shows cloudiness in the consistency with high concentrations and other samples are watery with low concentrations. could you please give me the reason?
    P.S, I have run the experiment at the same time!

    • Hi Hany, this article was written by the scientists at EpiGentek. I’m sure their tech support team can assist you with your troubleshooting question: https://www.epigentek.com/catalog/contact_us.php

      • Hany

        Hi B. Kirkpatrick, thank you for your help and I sent them an email. Hope they will provide me a solution!

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