Immunoprecipitation for endogenous HDAC1

Lysate from Daxx ^−/− and Daxx ^−/−;WT MEFs was prepared by resuspension in a total of 1.5 ml lysis buffer (20 mM HEPES-KOH pH 7.9, 300 mM KCl, 1 mM EDTA, 0.12% Triton X-100, 2 mM 2-mercaptoethanol, 0.4 mM PMSF, 2× protease inhibitor cocktail), followed by douncing and separation of the insoluble fraction by centrifugation. Per sample 50 µl of Dynabeads M-280 (anti-rabbit, Life Technologies) were washed briefly with PBS + 0.01% NP-40, then incubated with 5 µg of primary antibody (anti-HDAC1 or IgG control) for 1 h in PBS supplemented with 50 µg ml⁻¹ BSA. Beads were washed and then incubated with the prepare lysates for 3 h at 4 °C. Beads were washed three times with wash buffer (20 mM HEPES pH 7.9, 250 mM KCl, 1 mM EDTA, 0.12% Triton-X100, 0.4 mM PMSF) for 2 min at 4 °C. Immunoprecipitated proteins were eluted by incubation with Elution buffer (70 mM glycine pH 2.5, 150 mM NaCl) for 5 min at RT.