Follow the nice protocols from Abcam I copied here:

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 10 7 cells per ml.

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

4. Centrifuge at 6,500 x g for 10 minutes 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 minutes 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.

In my hands this is working very well and quick. You can stopp the reaction after over night in HCl by addition of 1/5 vol (of the HCl solution) of 1 M NaOH before centrifugation that you will have a normal pH. In case you want to determine the protein concentration via Bradford please you the composition of the final solution as background. Good luck!

Follow the nice protocols from Abcam I copied here:

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 10 7 cells per ml.

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

4. Centrifuge at 6,500 x g for 10 minutes 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 minutes 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.

In my hands this is working very well and quick. You can stopp the reaction after over night in HCl by addition of 1/5 vol (of the HCl solution) of 1 M NaOH before centrifugation that you will have a normal pH. In case you want to determine the protein concentration via Bradford please you the composition of the final solution as background. Good luck!

Dear Nicole Forster,

1. How long i can store Triton extraction buffer (TEB)? Is it necessary to use freshly prepared?

2. Also, can I use 5 X 10^5 cells/ml for histone extraction will it work for me?? as I am working on primary cell line. I wont be having enough cells.

Thanks

Akshata

Thanks Nicole. Can any one suggest protocol for isolation of histones from mesenchymal stem cells as we have tried above mentioned protocol which didn't work for hMSCs.

Thanks