Histone extraction from S.cerevisiae - (Jul/12/2013 )
I`ve found a protocol for histone extraction from S.cerevisia cells, but I don`t understand why I do some of the steps. Would be great if someone can help:
-Cells are harvested and washed
-zymolase treatment in 1.2 M sorbitol, 20 mM HEPES, pH 7.4. Why the sorbitol?
-wash with 1.2 M sorbitol, 20mM PIPES, 1mM MgCl2 (pH 6,8). Again: why sorbitol? Why the shift in pH?
-lysis in 0.25 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1mM CaCl2, 15 mM MES pH 6.6, 0.8% Triton X-100, 30 mM Sodium Butyrate. Why sucrose and the salts? Why again pH shift?
-wash nuclei in the buffer above, then in 10 mM Tris pH 8.0, 0.5% NP-40, 75 mM NaCl, 30 mM sodium butyrate and in 10 mM Tris pH 8.0, 0.4 M NaCl, 30 mM sodium butyrate. Why these? Why not just continue washing in the buffer above.
-Extract in 0.4 N H2SO4. What exactly does the acid do?
Thanks a lot for any help,
This is a typical high salt extraction assay,check this paper