Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade

5 product images

• 

  ChIP - Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

  Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 μg of chromatin, 2 μg of ab8898 (blue), and 20 μl of protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

• 

  Western blot - Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

  Lane 8 shows that Rabbit polyclonal to Histone H3 (tri methyl K9) is blocked by the addition of the immunizing peptide (Human Histone H3 (tri methyl K9) peptide ab1773). Cross-reactivity with Histone H3 peptide - tri methyl K27 (Human Histone H3 (tri methyl K27) peptide ab1782) is also shown in Lane 11.

  All lanes: Western blot - Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898) at 1 µg/mL

  All lanes: Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg

  Secondary

  All lanes: IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

  Performed under reducing conditions.

  Predicted band size: 15 kDa

  Observed band size: 17 kDa

• 

  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

  ab8898 staining Histone H3 (tri methyl K9) in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab8898 at 0.5µg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

  Also suitable in cells fixed with 4% paraformaldehyde (10 min).

  Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

  Histone H3 (tri methyl K9) Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-Histone H3 (tri methyl K9) antibody

  IHC image of ab8898 staining Histone H3 (tri methyl K9) in normal human colon 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 ab8898, 1/400 dilution, 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.

  *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• 

  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

  ICC/IF image of ab8898 stained HeLa 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 (ab8898, 0.1μg/ml) overnight at +4°C. The secondary antibody (green) was Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899, a goat anti-rabbit DyLight® 488 (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.