Rabbit anti histone h4

Whole cell protein extracts were prepared using SDS sample buffer (3 % SDS and 125 mM Tris-HCl, pH= 6.8) and heated at 95°C for 5 min. For immunoblotting analysis, 5 μg of total protein extracts were separated on 11 % SDS poly-acryl amide gels, transferred to polyvinylidene difluoride membranes (Millipore), blocked for 1 hour with 5 % non-fat dry milk in TBST buffer (137mM NaCl, 2.7mM KCl, 19mM Tris, 0.1% Tween). The following primary antibodies were used: rabbit- anti Gapdh (#5174S, Cell Signaling), mouse- anti H4K20me3 (#39672, Active Motif), rabbit- anti-histone H4 (#61200, Active Motif) and rabbit- anti Suv420h2 (#ab91224, Abcam). Washing was performed with TBS buffer containing 0.1 % Tween. Subsequently, binding of the secondary antibody (anti-rabbit IgG/anti-mouse IgG horseradish peroxidase-coupled) (Santa Cruz) diluted 1:10,000 in 5 % non-fat dry milk in TBST followed by detection with the Amersham ECL Prime Western Blot Detection Reagent (Ge Dharmacon) was carried out as described by the supplier. Chemo-luminescence was measured with an image acquisition system (Chemidoc Touch, Biorad). Measurements are given as means of 3 immuno-blots and representative blots are shown.