Dear Sir. Concerning your issue about the protocol for acid histone extraction from C. Elegant. Follow the protocols steps:

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.

I think the following below links may help you in your analysis:

http://www.abcam.com/protocols/histone-extraction-protocol-for-western-blot

https://theses.cz/id/murerd/downloadPraceContent_adipIdno_9750

Thanks

thank you very much Islam Eldin for your reply.

first of all sorry for the typo in my previous posting; it's C. Elgelans not Elegant (du to the automatic correction!)

The difficulty in my case is that I'm studying C Elegans not mammalian cells (see the attached figure. right lane = mESC histones; middle lane =worms extract). As I said I compared one protocol and it worked beautifully with mouse ESC but not with worms. the critical step is to get the nuclei and because they have a tough cuticle which necessitates to grind them and resuspend the powder in the nuclei isolation buffer. I think somewhere form then to the acid extraction and TCA precipitation I loose my histones.

I'll keep trying!

Thank you again