IHC image of ab130740 staining Histone H3 (symmetric di methyl R8) 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 ab130740, 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.
IHC image of Histone H3 (symmetric di methyl R8) staining in Human Skin formalin fixed paraffin embedded tissue section, performed on a Leica Bond 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 ab130740, 1µg/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.
Histone H3 was immunoprecipitated using 0.5mg Calf thymus histone whole cell extract, 5µg of Rabbit polyclonal to Histone H3 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).The antibody was incubated under agitation with Protein G beads for 10min, Calf thymus histone whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab130740.Secondary: Clean-Blot IP Detection Reagent (HRP) at 1/500 dilution.Band: 15kDa; Histone H3
Anti-Histone H3 (symmetric di methyl R8) antibody (ab130740) at 1 µg/ml + Calf Thymus Histone Preparation Nuclear Lysate at 0.25 µgSecondaryGoat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
All batches of ab130740 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 - symmetric di methyl R8 peptide (ab154299), indicating that this antibody specifically recognises the Histone H3 - symmetric di methyl R8 modification.ab154299 - Histone H3 - symmetric di methyl R8ab154295 - Histone H3 - asymmetric di methyl R8ab154298 - Histone H3 - mono methyl R8ab7228 - Histone H3 - unmodifiedab154465 - Histone H3 - symmetric di methyl R2ab154466 - Histone H3 - asymmetric di methyl R2ab154425 - Histone H3 - symmetric di methyl R17ab16935 - Histone H3 - asymmetric di methyl R17ab154427 - Histone H3 - symmetric di methyl R26ab2854 - Histone H3 - asymmetric di methyl R26
ICC/IF image of ab130740 stained HepG2 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 ab130740 at 1µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 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.