All lanes : Anti-Histone H3 (tri methyl K27, phospho S28) antibody (ab108268) at 1 µg/mlLane 1 : HeLa Histone Preparation Nuclear Lysate at 10 µgLane 2 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µgLane 3 : HeLa Histone Preparation Nuclear Lysate at 10 µg with Human Histone H3 (tri methyl K27, phospho S28) peptide (ab173750) at 1 µg/mlLane 4 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg with Human Histone H3 (tri methyl K27, phospho S28) peptide (ab173750) at 1 µg/mlLane 5 : HeLa Histone Preparation Nuclear Lysate at 10 µg with Human Histone H3 peptide (ab173779) at 1 µg/mlLane 6 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg with Human Histone H3 peptide (ab173779) at 1 µg/mlLane 7 : HeLa Histone Preparation Nuclear Lysate at 10 µg with Human Histone H3 (tri methyl K27) peptide (ab1782) at 1 µg/mlLane 8 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg with Human Histone H3 (tri methyl K27) peptide (ab1782) at 1 µg/mlLane 9 : HeLa Histone
ICC/IF Peptide competition assay performed on Methanol fixed HeLa cells. The cells were 100% Methanol fixed for 5 minutes at Room temperature and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-TWEEN for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The following peptides were chosen and incubated with ab108268 for 1 hour at room temperature, (non-mod block ab187169, Single modified block (tri-methyl-k27) ab1782, Single modified block (phosphor-S28) ab14793, Double modified block (K27 + S28) ab173822. ab108268 was used at 1ug/ml. Peptides were incubated at twice the concentration of the primary antibody. The cells were then incubated with either the peptide-competed antibody or the control antibody without peptide overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat anti-Rabbit (ab150081) IgG (H+L) preadsorbed used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 5