All lanes : Anti-Histone H2B (acetyl K11) antibody (ab40975) at 1 µg/mlLane 1 : Calf Thymus Histone Preparation Nuclear Lysate Lane 2 : Histone H2B Human recombinantLane 3 : Histone H3.1 Human recombinantLane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H2B (acetyl K11) peptide (ab41888) at 1 µg/mlLane 5 : Histone H2B Human recombinant with Human Histone H2B (acetyl K11) peptide (ab41888) at 1 µg/mlLane 6 : Histone H3.1 Human recombinant with Human Histone H2B (acetyl K11) peptide (ab41888) at 1 µg/mlLane 7 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H2B peptide (ab35791) at 1 µg/mlLane 8 : Histone H2B Human recombinant with Human Histone H2B peptide (ab35791) at 1 µg/mlLane 9 : Histone H3.1 Human recombinant with Human Histone H2B peptide (ab35791) at 1 µg/mlLysates/proteins at 10 µg per lane.SecondaryGoat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilutionPerformed under reducing conditions.
ICC/IF image of ab40975 stained human HeLa cells. The cells were methanol fixed (5 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab40975, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive IF result in HEK 293, HepG2 and MCF7 cells.
IHC image of ab40975 staining in cervix formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab40975, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.