Bis tris nupage novex

To extract histone proteins, embryos dissected at the indicated embryonic days, or about 5 x10 6 cells (were lysed with 100 μl acidic extraction buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT and 0.2 M HCl) freshly complemented with 1× Proteinase Inhibitor Cocktail (Roche) and 10 mM N-ethylmaleimide (Sigma-Aldrich).
HCl was added to a final concentration of 0.2 M and incubated on an end-to-end rotator for 2 hours at 4°C. Following the incubation, cell extract was centrifuged at 20 800 x g for 10 min at 4°C, to pellet the acid insoluble material. A solution of 2 M Tris-HCl (pH 8.8) was added to neutralize the supernatant of the acidic extraction. Ten μl of the supernatant, containing histone proteins, were run on 4-12% gels (Bis-tris NuPAGE Novex, Life Technologies), then proteins were transferred and western blot assays were carried out by using standard methods. The following antibodies were used: anti-H3 (Abcam #ab1791) anti-H4 (Invitrogen 3HH4-4G8), anti-H2Bub1 (Cell Signaling Technology, #5546), anti-H3K4me3 (Abcam ab8580), anti-H3K9ac (Merck-Millipore #07-352), Peroxidase AffiniPure F(ab') Fragment Goat Anti-Mouse IgG, Fcγ fragment specific (Jackson ImmunoResearch #115-036-071) and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch #115-035-144). Protein levels were quantified by ImageJ.