IHC image of ab47503 staining Histone H3 (mono methyl R17) in human kidney formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. 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 ab130283, 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. No primary antibody was used in the negative control (shown on the inset).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.
ICC/IF image of ab130283 stained MCF-7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab130283 at 1µg/ml overnight at +4°C. The secondary antibody (green) was a goat anti-rabbit DyLight® 488 (ab96899) IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
All batches of ab130283 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 mono methyl R17 peptide (ab154424), indicating that this antibody specifically recognises the Histone H3 mono methyl R17 modification.ab154424 - Histone H3 mono methyl R17ab17163 - Histone H3 unmodifiedab154425 - Histone H3 symmetric di methyl R17ab16935 - Histone H3 asymmetric di methyl R17ab1775 - Histone H3 mono methyl R2ab154298 - Histone H3 mono methyl R8ab154426 - Histone H3 mono methyl R26
All lanes : Anti-Histone H3 (mono methyl R17) antibody (ab130283) at 1/250 dilutionLane 1 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µgLane 2 : HeLa Histone Preparation Nuclear Lysate at 20 µgSecondaryGoat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.