Histone Extraction Protocol

Gozani Lab 4-25-05

Histone extraction protocol

1. Harvest cells and wash twice with ice-cold PBS. PBS can be supplemented with 5mM Sodium Butyrate to retain levels of histone acetylation.

2. Resuspend cells in Triton Extraction Buffer (TEB: PBS containing 0.5% Triton X 100

3. (v/v), 2mM phenylmethylsulfonyl fluoride (PMSF), 0.02% (v/v) NaN3) at a cell density of 10^7 cells ml^-1.

4. Lyse cells on ice for 10 minutes with gentle stirring.

5. Centrifuge at 2000rpm for 10 minutes at 4C. Remove and discard the supernatant.

6. Wash the cells in half the volume of TEB and centrifuge as in Step 5.

7. Resuspend the pellet in 0.2N HCl at a cell density of 4×10^7cells ml-1.

8. Acid extract the histones over night at 4C.

9. Centrifuge samples at 2000rpm for 10 minutes at 4C.

10. 9. Remove the supernatant and determine protein concentration using the Bradford assay.

11. 10. Store aliquots at -20C.

H-Lysis solution: 0.25M sucrose 3 mM CaCl2. 1 mM Tris pH8.0 0.5% NP-40 Filter sterilize, store at 4oC.

H-Wash solution: 300 mM NaCl. 5 mM MgCl2. 5 mM DTT 0.5% NP-40

H-Extraction solution: 0.5 M HCl 10% glycerol 0.1 M 2-mercaptoethylamine-HCl.