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.
- 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.
- 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.
- Centrifuge at 12,000 rpm for 5 minutes at 4°C and remove the supernatant fraction to a new vial.
- Add 8 volumes (approx. 0.6 ml/107 cells or 200 mg tissues) of acetone and leave at –20°C overnight.
- 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).
- 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.