All lanes : Anti-Histone H2B (acetyl K16) antibody - ChIP Grade (ab40977) at 1 µg/mlLane 1 : HeLa Histone Preparation Nuclear LysateLane 2 : HeLa Histone Preparation Nuclear Lysate - Butyrated treated Lane 3 : HeLa Histone Preparation Nuclear Lysate with Human Histone H2B (acetyl K16) peptide (ab41879) at 1 µg/mlLane 4 : HeLa Histone Preparation Nuclear Lysate - Butyrated treated with Human Histone H2B (acetyl K16) peptide (ab41879) at 1 µg/mlLane 5 : HeLa Histone Preparation Nuclear Lysate with Human Histone H2B peptide (ab35793) at 1 µg/mlLane 6 : HeLa Histone Preparation Nuclear Lysate - Butyrated treated with Human Histone H2B peptide (ab35793) at 1 µg/mlLysates/proteins at 2.5 µg per lane.SecondaryHRP Conjugated Goat Anti-Rabbit IgG at 1/3000 dilutionPerformed under reducing conditions.
ICC/IF image of ab40977 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab40977, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and 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).
IHC image of ab40977 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 ab40977, 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.