Anti-Histone H3 (di methyl K27) antibody - ChIP Grade

5 product images

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  This image is courtesy of an anonymous customer review.

  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K27) antibody - ChIP Grade (ab24684)

  Immunocytochemistry/ Immunofluorescence analysis of IMR32, Human Neuroblatoma cells labeling Histone H3 (di methyl K27) with ab24684 at 1/100 dilution. The cells were fixed with paraformaldehyde and permeabilized with 0.3% triton X 100. The cells were blocked with 1% BSA for 1 hour at 25°C, followed by incubation with primary antibody in 1% BSA containing TBST for 16 hours at 4°C. A Goat Anti Rabbit-IgG Cy3^® was used as the secondary antibody at 1/1000 dilution.

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  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K27) antibody - ChIP Grade (ab24684)

  ab24684 staining Histone H3 (di methyl K27) in HeLa cells. The cells were fixed with 100% Methanol (5min), 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 ab24684 at 1μg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control, at 1/1000 dilution. 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 (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 pseudocolor red). Nuclear DNA was labelled with DAPI (shown in blue).
  

  Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K27) antibody - ChIP Grade (ab24684)

  IHC image of Histone H3 (di methyl K27) staining in Human normal skin 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 ab24684, 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.
  

  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.

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  Western blot - Anti-Histone H3 (di methyl K27) antibody - ChIP Grade (ab24684)

  The peptide competition assay shows that this antibody is very specific for Histone H3 di-methylated at K27. It does not recognise Histone H3 mono

  , di

  , or tri methylated at K4

  , Histone H3 mono

  , di

  , or tri methylated at K9

  , or Histone H3 mono

  , or tri methylated at K27.

  All lanes: Western blot - Anti-Histone H3 (di methyl K27) antibody - ChIP Grade (ab24684) at 1 µg/mL

  All lanes: Calf thymus histone lysate

  Secondary

  All lanes: Goat polyclonal to Rabbit IgG H&L (HRP) at 1/5000 dilution

  Predicted band size: 15 kDa

  Observed band size: 17 kDa

  Exposure time: 5s

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  ChIP - Anti-Histone H3 (di methyl K27) antibody - ChIP Grade (ab24684)

  Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25μg of chromatin, 2μg of ab24684 (blue), and 20μl 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.